IP-10/Mig receptor designated CXCR3, antibodies, nucleic acids, and methods of use therefor

ABSTRACT

The present invention relates to proteins or polypeptides, referred to herein as isolated and/or recombinant mammalian (e.g., human) IP-10/Mig receptor proteins designated CXC Chemokine Receptor 3 (CXCR3) and variants thereof, including those characterized by selective binding of one or more chemokines (e.g., IP-10 and/or Mig), and/or the ability to induce a cellular response (e.g., chemotaxis, exocytosis). Antibodies reactive with CXCR3 receptors can be produced using the proteins or variants thereof or host cells comprising same as immunogen.  
     Another aspect of the invention relates to isolated and/or recombinant nucleic acids encoding a mammalian (e.g., human) CXCR3 protein and variants thereof, including antisense nucleic acid, recombinant nucleic acid constructs, such as plasmids or retroviral vectors, comprising a nucleic acid which encodes a protein of the present invention or variant thereof, and to host cells comprising a nucleic acid or construct, useful in the production of recombinant proteins. Also encompassed are methods of identifying ligands, and inhibitors (e.g., antagonists) or promoters (e.g., agonists) of receptor function, including methods in which host cells comprising a nucleic acid encoding a CXCR3 or variant thereof are used in an assay to identify and assess the efficacy of ligands, inhibitors or promoters. Inhibitors and promoters of receptor function can be used to modulate receptor activity, permitting selective inhibition of lymphocyte function, particularly of effector cells such as activated T lymphocytes and NK cells for therapeutic purposes.

RELATED APPLICATIONS

[0001] This application is a division of U.S. application Ser. No.09/663,702, filed Sep. 15, 2000, which is a continuation of U.S.application Ser. No. 08/829,839, filed Mar. 31, 1997 (now U.S. Pat. No.6,184,358 B1); and a continuation-in-part of U.S. application Ser. No.09/663,799, filed Sep. 15, 2000, which is a continuation of U.S.application Ser. No. 08/709,838, filed Sep. 10, 1996 (now U.S. Pat. No.6,140,064), the teachings of each of these applications are incorporatedherein by reference in their entirety.

DESCRIPTION BACKGROUND

[0002] Chemokines constitute a family of small cytokines that areproduced in inflammation and regulate leukocyte recruitment (BaggioliniM. et al., Adv. Immunol. 55: 97-179 (1994); Springer, T. A., Annu. Rev.Physiol. 57: 827-872 (1995); and Schall, T. J. and K. B. Bacon, Curr.Opin. Immunol. 6: 865-873 (1994)). Chemokines are capable of selectivelyinducing chemotaxis of the formed elements of the blood (other than redblood cells), including leukocytes such as neutrophils, monocytes,macrophages, eosinophils, basophils, mast cells, and lymphocytes, suchas T cells and B cells. In addition to stimulating chemotaxis, otherchanges can be selectively induced by chemokines in responsive cells,including changes in cell shape, transient rises in the concentration ofintracellular free calcium ions ([Ca²⁺]_(i)), granule exocytosis,integrin upregulation, formation of bioactive lipids (e.g.,leukotrienes) and respiratory burst, associated with leukocyteactivation. Thus, the chemokines are early triggers of the inflammatoryresponse, causing inflammatory mediator release, chemotaxis andextravasation to sites of infection or inflammation.

[0003] Two subfamilies of chemokines, designated as CXC and CCchemokines, are distinguished by the arrangement of the first two offour conserved cysteine residues, which are either separated by oneamino acid (as in CXC chemokines IL-8, γIP-10, Mig, PF4, ENA-78, GCP-2,GROα, GROβ, GROγ, NAP-2, NAP-4) or are adjacent residues (as in CCchemokines MIP-1α, MIP-1β, RANTES, MCP-1, MCP-2, MCP-3, I-309). Most CXCchemokines attract neutrophil leukocytes. For example, the CXCchemokines interleukin 8 (IL-8), platelet factor 4 (PF4), andneutrophil-activating peptide 2 (NAP-2) are potent chemoattractants andactivators of neutrophils. The CXC chemokines designated Mig (monokineinduced by gamma interferon) and IP-10 (γIP-10, interferon-gammainducible 10 kDa protein) are particularly active in inducing chemotaxisof activated peripheral blood lymphocytes. CC chemokines are generallyless selective and can attract a variety of leukocyte cell types,including monocytes, eosinophils, basophils, T lymphocytes and naturalkiller cells. CC chomokines such as human monocyte chemotactic proteins1-3 (MCP-1, MCP-2 and MCP-3), RANTES (Regulated on Activation, Normal TExpressed and Secreted), and the macrophage inflammatory proteins 1α and1β (MIP-1α and MIP-1β) have been characterized as chemoattractants andactivators of monocytes or lymphocytes, but do not appear to bechemoattractants for neutrophils.

[0004] CC and CXC chemokines act through receptors which belong to asuperfamily of seven transmembrane spanning G protein-coupled receptors(Murphy, P. M., Annu. Rev. Immunol., 12: 593-633 (1994); Gerard, C. andN. P. Gerard, Curr. Opin. Immunol., 6: 140-145 (1994)). This family ofG-protein coupled (serpentine) receptors comprises a large group ofintegral membrane proteins, containing seven transmembrane-spanningregions. The receptors are coupled to G proteins, which areheterotrimeric regulatory proteins capable of binding GTP and mediatingsignal transduction from coupled receptors, for example, by theproduction of intracellular mediators.

[0005] The chemokine receptors can be divided into two groups: CCchemokine receptors 1 through 5 (CCR1-5), which bind CC chemokines, andCXC chemokine receptors 1 through 4 (CXCR1-4), which bind CXCchemokines. In general, the CC chemokine receptors occur on severaltypes of leukocytes, and are important for the migration of monocytes,eosinophils, basophils, and T cells (Qin, S., et al., Eur. J. Immunol.,26:640-647 (1996); Carr, M. W., et al., Proc. Natl. Acad. Sci. USA,91(9):3652-3656 (1994); Taub, D. D., et al., J. Clin. Invest.,95(3):1370-1376 (1995); Neote, K. et al., Cell, 72: 415-425 (1993); Gao,J. -L. et al., J. Exp. Med., 177: 1421-1427 (1993); Charo, I. F. et al.,Proc. Natl. Acad. Sci. USA, 91: 2752-2756 (1994); Myers, S. J., et al.,J. Biol. Chem., 270: 5786-5792 (1995); Combadiere, C. et al., J. Biol.Chem., 270 (27): 16491-16494 (1995); and Correction, J. Biol. Chem.,270: 30235 (1995); Ponath, P. D. et al., J. Exp. Med., 183: 2437-2448(1996); and Daugherty, B. L. et al., J. Exp. Med., 183: 2349-2354(1996); Power, C. A. et al., 1995, J. Biol. Chem., 270: 19495-19500(1995); Hoogewerf, A. J. et al., Biochem. Biophys. Res. Commun., 218:337-343 (1996); Samson, M. et al., Biochemistry, 35: 3362-3367 (1996)).In contrast, the two IL-8 receptors, CXCR1 and CXCR2, are largelyrestricted to neutrophils and are important for the migration ofneutrophils (Baggiolini, M., et al., Adv. Immunol., 55:97-179 (1994)).The IL-8 receptors, CXCR1 (IL-8R1, interleukin-8 receptor type 1;Holmes, W. E. et al., Science, 253: 1278-1280 (1991)) and CXCR2 (IL-8R2,interleukin-8 receptor type 2; Murphy, P. M. and H. L. Tiffany, Science,253: 1280-1283 (1991)) recognize the NH2-terminal Glu-Leu-Arg (ELR)motif, an essential binding epitope observed in CXC chemokines thatinduce neutrophil chemotaxis (Clark-Lewis, I. et al., J. Biol. Chem.,266: 23128-23134 (1991); Hebert, C. A. et al., J. Biol. Chem., 266:18989-18994 (1991); and Clark-Lewis, I. et al., Proc. Natl. Acad. Sci.USA, 90: 3574-3577 (1993)).

[0006] In contrast to monocytes and granulocytes, lymphocyte responsesto chemokines are not well understood. Notably, none of the receptors ofknown specificity appear to be restricted to lymphocytes and thechemokines that recognize these receptors cannot, therefore, account forevents such as the selective recruitment of T lymphocytes that isobserved in T cell-mediated inflammatory conditions. Moreover, althougha number of proteins with significant sequence similarity and similartissue and leukocyte subpopulation distribution to known chemokinereceptors have been identified and cloned, the ligands for thesereceptors remain undefined. Thus, these proteins are referred to asorphan receptors. The characterization of the ligand(s) of a receptor,is essential to an understanding of the interaction of chemokines withtheir target cells, the events stimulated by this interaction, includingchemotaxis and cellular activation of leukocytes, and the development oftherapies based upon modulation of receptor function.

SUMMARY OF THE INVENTION

[0007] The present invention relates to proteins or polypeptides,referred to herein as isolated and/or recombinant mammalian (e.g., aprimate such as a human) IP-10/Mig receptor proteins designated CXCChemokine Receptor 3 (CXCR3) and variants thereof. Recombinant CXCR3proteins and variants can be produced in host cells as described herein.In one embodiment, a CXCR3 protein or variant thereof is characterizedby selective binding (e.g., high affinity binding) of one or morechemokines, such as IP-10 and/or Mig, and/or the ability to induce a(one or more) cellular response(s) (e.g., chemotaxis, exocytosis,release of one or more inflammatory mediators).

[0008] Another aspect of the present invention relates to isolatedand/or recombinant nucleic acids which encode a mammalian (e.g., aprimate such as a human) CXCR3 protein or variant thereof. The inventionfurther relates to recombinant nucleic acid constructs, such as plasmidsor retroviral vectors, which contain a nucleic acid which encodes aprotein of the present invention or a variant thereof. The nucleic acidsand constructs can be used to produce recombinant receptor proteins andhost cells comprising a construct. In another embodiment, the nucleicacid encodes an antisense nucleic acid which can hybridize with a secondnucleic acid encoding a CXCR3 protein and which, when introduced intocells, can inhibit the expression of receptor.

[0009] The invention further relates to antibodies reactive with CXCR3receptors, which can be produced using the proteins or variants thereof(e.g., a peptide) or cells expressing receptor protein or variant asimmunogen, for example. Such antibodies or fragments thereof are usefulin therapeutic, diagnostic and research applications, including thepurification and study of the receptor proteins, identification of cellsexpressing surface receptor, and sorting or counting of cells. Thus, thepresent invention encompasses use of an antibody or fragment thereofdescribed herein (e.g., mAb 1C6 or an antigen-binding fragment thereof)in therapy (including prophylaxis) or diagnosis, and use of suchantibodies or fragments for the manufacture of a medicament for use intreatment of diseases or conditions as described herein.

[0010] Also encompassed by the present invention are methods ofidentifying ligands of the receptor, inhibitors (e.g., antagonists) orpromoters (e.g., agonists) of receptor function. In one embodiment,suitable host cells which have been engineered to express a receptorprotein or variant encoded by a nucleic acid introduced into said cellsare used in an assay to identify and assess the efficacy of ligands,inhibitors or promoters of receptor function. Such cells are also usefulin assessing the function of the expressed receptor protein orpolypeptide.

[0011] According to the present invention, ligands, inhibitors andpromoters of receptor function can be identified in a suitable assay,and further assessed for therapeutic effect. Inhibitors of receptorfunction can be used to inhibit (reduce or prevent) receptor activity,and ligands and/or promoters can be used to induce (trigger or enhance)normal receptor function where indicated. Thus, the present inventionprovides a method of treating inflammatory diseases including autoimmunedisease and graft rejection, comprising administering an inhibitor ofreceptor function to an individual (e.g., a mammal). The presentinvention further provides a method of stimulating receptor function byadministering a ligand or promoter to an individual, providing a newapproach to selective stimulation of leukocyte function, which isuseful, for example, in the treatment of infectious diseases and cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is an illustration of the nucleotide sequence determinedfrom the 1670 bp insert of a cDNA encoding a human IP-10/Mig receptordesignated CXCR3, which was isolated from a human CD4⁺T cell (KT30) cDNAlibrary (SEQ ID NO:1). An open reading frame (69-1175) encodes apredicted protein of 368 amino acids (SEQ ID NO:2). A putative poly-Asignal and poly A site are located at positions 1534-1539 and at1624-1670, respectively.

[0013]FIG. 2 is an illustration of the conceptual translation of theopen reading frame of the sequence in FIG. 1, which encodes a humanIP-10/Mig receptor (SEQ ID NO:2). Arrowheads indicate potential N-linkedglycosylation sites and horizontal lines indicate the location ofputative transmembrane domains (TM1-TM7).

[0014] FIGS. 3A-3C are graphs illustrating the responses induced byIP-10 and Mig in stably transfected cells expressing IP-10/MigR. FIG. 3Ais a graph illustrating the concentration dependent [Ca²⁺]_(i) changesin IP-10/MigR-transfected 300-19 cells. IP-10 or Mig were each added at1, 10, and 100 nM to Fura-2/AM loaded cells (arrowhead), and[Ca²⁺]_(i)-dependent fluorescence changes were recorded. Non-transfectedcells (lower tracings) were stimulated with IP-10 or Mig at 100 nM underidentical conditions.

[0015]FIG. 3B is a graph illustrating the results of studies assessingreceptor desensitization and cross-desensitization, in which IP-10/MigRexpressing 300-19 cells were sequentially stimulated with 100 nM IP-10or Mig, and with IP-10 followed by Mig or vice versa, and fluorescencechanges were recorded.

[0016]FIG. 3C is a graph illustrating the chemotaxis of IP-10/MigRexpressing Jurkat cells stimulated with IP-10 (filled circles) or Mig(filled squares). The lower panel shows the response of non-transfectedJurkat cells when stimulated with increasing amounts of IP-10 (opencircles) or Mig (open squares). Mean numbers (+SD) of migrating cellsper five high power fields are presented.

[0017] FIGS. 4A-4B are graphs illustrating the responses of peripheralblood lymphocytes (PBL) to IP-10 and Mig. Freshly isolated PBL fromdonor blood buffy coats were used as such (lower tracings and opensymbols), or were used after culturing for 10 days in the presence ofIL-2 (400 U/ml) (upper tracings and closed symbols). FIG. 4A is a graphillustrating [Ca²⁺]_(i) changes induced by IP-10 or Mig. IP-10 or Migwere each added at 1, 10, and 100 nM to Fura-2/AM loaded cultured cells(arrowhead), and [Ca²⁺]_(i)-dependent fluorescence changes were recorded(upper tracings). Freshly isolated cells (lower tracings) werestimulated with IP-10 or Mig at 100 nM under the same conditions. FIG.4B is a graph illustrating chemotaxis of PBL in response to increasingconcentrations of IP-10 (filled circles) or Mig (filled squares) (meannumbers (±SD) of migrating cells per five high power fields arepresented).

[0018] FIGS. 5A-5B are graphs illustrating the binding of radiolabeledIP-10 to either L1.2 cells transfected with CXCR3 DNA (FIG. 5A) and toactivated T cells (FIG. 5B). Cells were incubated with 0.05 nM¹²⁵I-labeled IP-10 in the presence of increasing concentrations ofunlabeled IP-10. Scatchard analysis (inset) indicated 37,000 receptorsper cell (Kd of 614 pM) for CXCR3 L1.2 transfectants, and 17,000receptors per cell (Kd of 156 pM) for CD3 blasts.

[0019]FIG. 6 is an illustration of the specificity of anti-CXCR3antibody 1C6 as assessed by staining of stable L1.2 transfectantsexpressing either CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3 orCXCR4 by anti-CXCR3 peptide mAb 1C6. Negative control staining for allthe L1.2 transfectants (not shown) resembled the staining shown for 1C6on untransfected L1.2 cells (L1.2 wt).

[0020] FIGS. 7A-7C are fluorescence histograms illustrating theexpression of CXCR3 on neutrophils (FIG. 7A), lymphocytes (FIG. 7B), andactivated T cells (FIG. 7C) Leukocyte subsets were identified in wholeblood by their forward angle and side scatter, and were gatedaccordingly. To generate CD3 blasts, PBMC were activated with anti-CD3mAb for 3 days, and were then maintained in media containing IL-2 for 7days. In each plot, the blackened profile represents staining withanti-CXCR3 mAb 1C6, and the unfilled profile represent staining with anisotype-matched control mAb.

[0021]FIG. 8 is a series of plots illustrating CXCR3 expression onpopulations of blood lymphocytes. A two color staining protocol was usedto assess expression of CXCR3 on T cells (CD3), B cells (CD20), and NKcells (CD56).

[0022]FIG. 9 is a series of plots illustrating CXCR3 expression versusvarious markers on the CD3+subset of blood lymphocytes, as analyzed bythree color analysis of immunofluorescence. Anti-CD3 Cy-Chrome was usedto stain T cells, and these cells were gated electronically foranalysis. Quadrants were set according to the staining of control mkbs.The staining shown was representative of five donors analyzed.

[0023]FIG. 10 is a histogram illustrating inhibition of IP-10- orMCP-1-mediated chemotaxis of activated T cells by a panel of anti-CXCR3mAbs. 1×10⁶ human CD3 blasts were placed in the top chamber of atranswell and chemokine (12.5 nM) was placed in the bottom chamber.Various anti-CXCR3 mAbs (in tissue culture supernatant, without FCS)were placed in the top well with cells at the beginning of the assay.After 1.5 hours the cells migrating to the bottom chamber were countedusing flow cytometry. The percentage inhibition of chemotaxis wascalculated using the number of cells that migrated in the absence of mAbas 1000. The results are representative of at least four separateexperiments.

[0024]FIG. 11 is a graph illustrating the inhibition of IP-10 mediatedchemotaxis by purified anti-CXCR3 mAb 1C6. Various concentrations of 1C6mAb were placed in the top well, and the assay was performed asdescribed for FIG. 10. mAb 1C6 inhibited 50% total chemotaxis at aconcentration of 856 ng/ml (IC₅₀ ⁼⁸⁵⁶ ng/ml).

[0025]FIG. 12 is a graph illustrating the inhibition of ¹²⁵I-IP-10binding to activated T cells by mAb 1C6. CD3 blasts were incubated with0.05 nM ¹²⁵I-Ip-10 in the presence of increasing concentrations of 1C6as indicated. After 60 minutes at room temperature, cell pellets werewashed and counted. Data was analyzed by KaleidaGraph, which gave anIC₅₀ of 0.16 μg/ml.

[0026] FIGS. 13A-13H illustrate the inhibition by mAb 1C6 of [Ca²⁺]_(i)by human T cells in response to IP-10, but not Mig. Anti-CD3 activated,IL-2-stimulated human T cells were labeled with Fura-2, and werestimulated sequentially with the indicated chemokines (FIGS. 13A-13B),or with mAb followed 40 seconds later by the indicated chemokine (FIGS.13C-13H). [Ca²⁺]_(i) fluorescence changes were recorded using aspectrofluorimeter. The tracings were representative of five separateexperiments. Antibody was used at a final concentration of either 50μg/ml (FIGS. 13C-13D); 25 μg/ml (FIG. 13E); 12.5 μg/ml (FIG. 13F); 6.125μg/ml (FIG. 13G); or 3.0625 μg/ml (FIG. 13H)I Chemokines were used at 2nM.

[0027] FIGS. 14A-14D are fluorescence histograms illustrating theresults of a flow cytometry analysis in which CXCR3-expressingtransfectants were stained with mAb 1C6 in the presence of P1 peptide(FIG. 14B), P2 peptide (FIG. 14C), P3 peptide (FIG. 14D), or in theabsence of peptide (FIG. 14A). In each plot, the profile defined by theheavy line represents mAb 1C6 staining, and the profile defined by thedotted line represents staining with an isotype-matched irrelevantcontrol mAb.

[0028]FIG. 15 is a histogram illustrating the percent inhibition ofbinding of radiolabeled IP-10 to CXCR3 transfectants by 40 nM coldIP-10, mAb 1C6, monoclonal antibodies raised against CXCR3 transfectants(2F8, 3A12, 3E2, 4B4, 4D2, 5B12, 7B8, or 8D5), or by anti-CXCR2 mAb.

DETAILED DESCRIPTION OF THE INVENTION

[0029] As described herein, a nucleic acid encoding a novel chemokinereceptor that is selective for the CXC chemokines IP-10 and Mig wascloned and characterized. The clone, which was isolated from a humanCD4⁺ T cell library, was not detected in monocyte- orgranulocyte-derived cDNA libraries. Sequence analysis of the clonerevealed an open reading frame of 1104 base pairs (FIG. 1, SEQ ID NO:1),encoding a predicted protein of 368 amino acids with a predictedmolecular mass of 40,659 daltons (FIG. 2, SEQ ID NO:2). The amino acidsequence includes seven putative transmembrane segments which arecharacteristic of G-protein coupled receptors and are found in otherchemoattractant receptors. Consistent with this observation, thereceptor mediates Ca²⁺ (calcium ion) mobilization and chemotaxis inresponse to IP-10 and Mig (Example 2). No significant response to theCXC chemokines IL-8, GROα, NAP-2 (neutrophil-activating protein-2),GCP-2 (granulocyte chemotactic protein-2), ENA78 (epithelial-derivedneutrophil-activating peptide 78), PF4 (platelet factor 4), or the CCchemokines MCP-1 (monocyte chemotactic protein-1), MCP-2, MCP-3, MCP-4,MIP-1α (macrophage inflammatory protein-1α), MIP-10, RANTES (regulatedon activation, normal T cell expressed and secreted), I309, eotaxin orlymphotactin was detected under similar conditions.

[0030] The restricted expression of human CXCR3 in activated Tlymphocytes and the ligand selectivity of the receptor for IP-10 and Migare noteworthy. The human receptor is highly expressed in IL-2 activatedT lymphocytes, but was not detected in resting T lymphocytes, Blymphocytes, monocytes or granulocytes under the conditions used inExample 2. Additional studies of receptor distribution indicate that itis mostly CD3+ cells that express CXCR3, including cells which areCD95+, CD45RO+, and CD45RA^(low), a phenotype consistent with previousactivation, although a proportion of CD20+ (B) cells and CD56+ (NK)cells also express this receptor. The selective expression in activatedT lymphocytes is of interest, because other receptors for chemokineswhich have been reported to attract lymphocytes (e.g., MCP-1, MCP-2,MCP-3, MIP-1α, MIP-1β and RANTES) are also found in granulocytes, suchas neutrophils, eosinophils and basophils, as well as monocytes. Theseresults suggest that the IP-10/Mig receptor designated CXCR3 is involvedin the selective recruitment of effector T cells.

[0031] The receptor recognizes two unusual CXC chemokines, designatedIP-10 and Mig. Although IP-10 and Mig both belong to the CXC subfamily,in contrast to IL-8 and other CXC chemokines which are potentchemoattractants for neutrophils, the primary targets of IP-10 and Migare lymphocytes, particularly effector cells such as activated orstimulated T lymphocytes and natural killer (NK) cells (Taub, D. D. etal., J. Exp. Med., 177: 18090-1814 (1993); Taub, D. D. et al., J.Immunol., 155: 3877-3888 (1995)). (NK cells are large granularlymphocytes, which lack a specific T cell receptor for antigenrecognition, but possess cytolytic activity against cells such as tumorcells and virally infected cells.) Consistently, IP-10 and Mig lack theELR motif, an essential binding epitope in those CXC chemokines thatefficiently induce neutropiil chemotaxis (Clark-Lewis, I. et al., J.Biol. Chem., 266: 23128-23134 (1991); Hebert, C. A. et al., J. Biol.Chem., 266: 18989-18994 (1991); and Clark-Lewis, I. et al., Proc. Natl.Acad. Sci. USA, 90: 3574-3577 (1993)). In addition, both recombinanthuman Mig and recombinant human IP-10 have been reported to inducecalcium flux in tumor infiltrating lymphocytes (TIL) (Liao, F. et al.,J. Exp. Med., 182: 1301-1314 (1995)). While IP-10 has been reported toinduce chemotaxis of monocytes in vitro (Taub, D. D. et al., J. Exp.Med., 177: 1809-1814 (1993), the receptor responsible has not beenidentified), human Mig appears highly selective, and does not show suchan effect (Liao, F. et al., J. Exp. Med., 182: 1301-1314 (1995)). IP-10expression is induced in a variety of tissues in inflammatory conditionssuch as psoriasis, fixed DRUG eruptions, cutaneous delayed-typehypersensitivity responses, tuberculoid leprosy, and in experimentalglomerulonephritis, and experimental allergic encephalomyelitis. IP-10also has a potent in vivo antitumor effect that is T cell dependent, isreported to be an inhibitor of angiogenesis in vivo, and can inducechemotaxis and degranulation of NK cells in vitro, suggesting a role asa mediator of NK cell recruitment and degranulation (in tumor celldestruction, for example) (Luster, A. D. and P. Leder, J. Exp. Med.,178: 1057-1065 (1993); Luster, A. D. et al., J. Exp. Med. 182: 219-231(1995); Angiolillo, A. L. et al., J. Exp. Med., 182: 155-162 (1995);Taub, D. D. et al., J. Immunol., 155: 3877-3888 (1995)). The expressionpatterns of IP-10 and Mig are also distinct in that expression of eachis induced by interferon-gamma (IFNγ), while the expression of IL-8 isdown-regulated by IFNγ (Luster, A. D. et al., Nature, 315: 672-676(1985); Farber, J. M., Proc. Natl. Acad. Sci. USA, 87: 5238-5242 (1990);Farber, J. M., Biochem. Biophys. Res. Commun., 192 (1): 223-230 (1993),Liao, F. et al., J. Exp. Med., 182: 1301-1314 (1995); Seitz, M. et al.,J. Clin. Invest., 87: 463-469 (1991); Caly, A. H. M. and H. Spits, J.Immunol., 147: 3823-3830 (1991)).

[0032] Chemokines have been recently recognized as the long-soughtmediators for the recruitment of lymphocytes. Several CC chemokines werefound to elicit lymphocyte chemotaxis (Loetscher, P. et al., FASEB J.,8: 1055-1060 (1994)), but they are also active on granulocytes andmonocytes (Uguccioni, M. et al., Eur. J. Immunol., 25: 64-68 (1995);Baggiolini, M. and C. A. Dahinden, Immunol. Today, 15: 127-133 (1994)).The situation is different for IP-10 and Mig, which are selective intheir action on lymphocytes, including activated T lymphocytes and NKcells, and which bind CXCR3, a receptor which does not recognizenumerous other chemokines and which displays a selective pattern ofexpression (Example 2, Example 5).

[0033] In view of these observations, it is reasonable to conclude thatthe formation of the characteristic infiltrates in inflammatory lesions,such as delayed-type hypersensitivity lesions, sites of viral infection,and certain tumors is a process mediated by via CXCR3 and regulated byCXCR3 expression. Lymphocytes, particularly T lymphocytes, bearing aCXCR3 receptor as a result of activation can be recruited intoinflammatory lesions, sites of infection, or tumors by IP-10 and/or Mig,which can be induced locally by interferon-gamma. Thus, CXCR3 plays arole in the selective recruitment of lymphocytes, particularly effectorcells such as activated or stimulated T lymphocytes.

[0034] Proteins and Peptides

[0035] The present invention relates to isolated and/or recombinant(including, e.g., essentially pure) proteins or polypeptides designatedmammalian CXCR3 proteins and variants thereof. In a preferredembodiment, the isolated and/or recombinant proteins of the presentinvention have at least one property, activity or functioncharacteristic of a mammalian CXCR3 protein (as defined herein), such asa binding activity (e.g., ligand, inhibitor and/or promoter binding), asignalling activity (e.g., activation of a mammalian G protein,induction of rapid and transient increase in the concentration ofcytosolic free calcium [Ca²⁺]_(i)), cellular response function (e.g.,stimulation of chemotaxis, exocytosis or inflammatory mediator releaseby leukocytes), and/or an immunological property as defined herein. Forexample, some proteins of the present invention can selectively bind toIP-10 and/or Mig, mediate cellular signalling and/or a response theretoin vitro and/or in vivo (e.g., calcium flux, chemotaxis and/ordegranulation especially of activated T lymphocytes). For example, asshown herein, a human CXCR3 protein, produced in mammalian cells byexpression of a cDNA clone, can selectively bind to CXC chemokines IP-10and/or Mig, and mediate signalling, and a cellular response (e.g.,chemotaxis). In one embodiment, proteins of the present invention canbind a CXC chemokine from the same or a different mammalian species(e.g., human IP-10, murine IP-10, human Mig, murine Mig) (human IP-10,Luster, A. D. et al., Nature, 315: 672-676 (1985); murine IP-10 (alsoreferred to as CRG-2), Vanguri, P. and J. M. Farber, J. Biol. Chem.,265: 15049 (1990) and Luster, A. D. and P. Leder, J. Exp. Med., 178:1057-1065 (1993); murine Mig, Farber, J. M., Proc. Natl. Acad. Sci. USA,87: 5238-5242 (1990); human Mig, Farber, J. M., Biochem. Biophys. Res.Commun., 192 (1): 223-230 (1993 and Liao, F. et al., J. Exp. Med., 182:1301-1314 (1995)).

[0036] Proteins or polypeptides referred to herein as “isolated” areproteins or polypeptides purified to a state beyond that in which theyexist in mammalian cells, and include proteins or polypeptides obtainedby methods described herein, similar methods or other suitable methods,including essentially pure proteins or polypeptides, proteins orpolypeptides produced by chemical synthesis (e.g., synthetic peptides),or by combinations of biological and chemical methods, and recombinantproteins or polypeptides which are isolated. The proteins can beobtained in an isolated state of at least about 50% by weight,preferably at least about 75% by weight, or in essentially pure form.Proteins or polypeptides referred to herein as “recombinant” areproteins or polypeptides produced by the expression of recombinantnucleic acids.

[0037] As used herein “mammalian CXCR3 protein” refers to naturallyoccurring or endogenous mammalian CXCR3 proteins and to proteins havingan amino acid sequence which is the same as that of a naturallyoccurring or endogenous corresponding mammalian CXCR3 protein (e.g.,recombinant proteins). Accordingly, as defined herein, the term“mammalian CXCR3 protein” includes mature protein, polymorphic orallelic variants, and other isoforms of mammalian CXCR3 (e.g., producedby alternative splicing or other cellular processes), and modified orunmodified forms of the foregoing (e.g., glycosylated, unglycosylated,phosphorylated or unphosphorylated CXCR3 proteins). Naturally occurringor endogenous mammalian CXCR3 proteins include wild type proteins suchas mature CXCR3, polymorphic or allelic variants and other isoformswhich occur naturally in mammals (e.g., humans, non-human primates).Such proteins can be recovered from a source which naturally producesmammalian CXCR3, for example. These proteins and mammalian CXCR3proteins having the same amino acid sequence as a naturally occurring orendogenous corresponding mammalian CXCR3, are referred to by the name ofthe corresponding mammal. For example, where the corresponding mammal isa human, the protein is designated as a human CXCR3 protein (e.g., arecombinant human CXCR3 produced in a suitable host cell).

[0038] “Functional variants” of mammalian CXCR3 proteins includefunctional fragments, functional mutant proteins, and/or functionalfusion proteins (e.g., produced via mutagenesis and/or recombinanttechniques). Generally, fragments or portions of mammalian CXCR3proteins encompassed by the present invention include those having adeletion (i.e., one or more deletions) of an amino acid (i.e., one ormore amino acids) relative to the mature mammalian CXCR3 protein (suchas N-terminal, C-terminal or internal deletions). Fragments or portionsin which only contiguous amino acids have been deleted or in whichnon-contiguous amino acids have been deleted relative to maturemammalian CXCR3 protein are also envisioned.

[0039] Generally, mutants or derivatives of mammalian CXCR3 proteins,encompassed by the present invention include natural or artificialvariants differing by the addition, deletion and/or substitution of oneor more contiguous or non-contiguous amino acid residues, or modifiedpolypeptides in which one or more residues is modified, and mutantscomprising one or more modified residues. Preferred mutants are naturalor artificial variants of mammalian CXCR3 proteins differing by theaddition, deletion and/or substitution of one or more contiguous ornon-contiguous amino acid residues. Such mutations can be in a conservedregion or nonconserved region (compared to other CXC and/or CC chemokinereceptors), extracellular, cytoplasmic, or transmembrane region, forexample.

[0040] A “functional fragment or portion”, “functional mutant” and/or“functional fusion protein” of a mammalian CXCR3 protein refers to anisolated and/or recombinant protein or oligopeptide which has at leastone property, activity or function characteristic of a mammalian CXCR3receptor (as defined herein), such as a binding activity (e.g., ligand,inhibitor and/or promoter binding), a signalling activity (e.g.,activation of a mammalian G protein, induction of rapid and transientincrease in the concentration of cytosolic free calcium [Ca²⁺]_(i)),cellular response function (e.g., stimulation of chemotaxis, exocytosisor inflammatory mediator release by leukocytes) and/or an immunologicalproperty as defined herein.

[0041] As used herein, a protein or polypeptide having “at least oneimmunological property” of a mammalian CXCR3 protein is one which (a) isbound by at least one antibody of a selected epitopic specificity whichbinds to a naturally occurring or endogenous mammalian CXCR3 protein orto a protein having the same amino acid seqence as the naturallyoccurring or endogenous mammalian CXCR3 protein (e.g., human CXCR3),and/or (b) is an immunogen capable of inducing the formation (e.g., whenconjugated to a suitable carrier) in a suitable animal of an antibody ofa selected epitopic specificity which binds to a naturally occurring orendogenous mammalian CXCR3 or to a protein having the same amino acidseqence as the naturally occurring or endogenous mammalian CXCR3. Forexample, a suitable fragment can cross-react with an antibody which israised against and/or reactive with isolated mammalian CXCR3.

[0042] Suitable fragments or mutants can be identified by screening. Forexample, the N-terminal, C-terminal, or internal regions of the proteincan be deleted in a step-wise fashion and the resulting protein orpolypeptide can be screened using a suitable assay, such as an assaydescribed herein (e.g., chemotaxis, calcium flux). Where the resultingprotein displays activity in the assay, the resulting protein(“fragment”) is functional. Information regarding the structure andfunction of mammalian G protein coupled receptors, including CXCchemokine and CC chemokine receptors, provides a basis for dividingmammalian CXCR3 proteins into functional domains (Murphy, P. M., Annu.Rev. Immunol., 12: 593-633 (1994) and Gerard, C. and N. P. Gerard, Curr.Opin. Immunol., 6: 140-145 (1994), and references cited therein).

[0043] The term variant also encompasses fusion proteins, comprising amammalian CXCR3 proteins (e.g., human CXCR3) as a first moiety, linkedto a second moiety not occurring in the mammalian CXCR3 as found innature. Thus, the second moiety can be an amino acid, oligopeptide orpolypeptide. The first moiety can be in an N-terminal location,C-terminal location or internal to the fusion protein. In oneembodiment, the fusion protein comprises an affinity ligand (e.g., anenzyme, an antigen, epitope tag) as the first moiety, and a secondmoiety comprising a linker sequence and human CXCR3 or portion thereof.

[0044] Examples of mammalian CXCR3 proteins include proteins encoded bya nucleic acid of the present invention, such as a protein having anamino acid sequence as set forth or substantially as set forth in FIG. 2(SEQ ID NO:2). In a preferred embodiment, a mammalian CXCR3 or variant(e.g., a variant including the extracellular N-terminal segment) has anamino acid sequence which is at least about 50% identical, morepreferably at least about 70% identical, and still more preferably atleast about 80% identical, to the protein shown in FIG. 2 (SEQ ID NO:2).

[0045] It will be appreciated that isolated and/or recombinant mammalianCXCR3 proteins and variants thereof can be modified, for example, byincorporation of or attachment (directly or indirectly (e.g., via alinker)) of a detectable label such as a radioisotope, spin label,antigen (e.g., epitope label such as a FLAG tag) or enzyme label,flourescent or chemiluminesent group and the like, and such modifiedforms are included within the scope of the invention.

[0046] Nucleic Acids, Constructs and Vectors

[0047] The present invention relates to isolated and/or recombinant(including, e.g., essentially pure) nucleic acids having sequences whichencode a mammalian (e.g., human) CXCR3 protein or variant thereof asdescribed herein. Nucleic acids referred to herein as “isolated” arenucleic acids separated away from the nucleic acids of the genomic DNAor cellular RNA of their source of origin (e.g., as it exists in cellsor in a mixture of nucleic acids such as a library), and may haveundergone further processing. “Isolated” nucleic acids include nucleicacids obtained by methods described herein, similar methods or othersuitable methods, including essentially pure nucleic acids, nucleicacids produced by chemical synthesis, by combinations of biological andchemical methods, and recombinant nucleic acids which are isolated.Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial recombination, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes. “Recombinant”nucleic acids are also those that result from recombination events thatoccur through the natural mechanisms of cells, but are selected forafter the introduction to the cells of nucleic acids designed to allowand make probable a desired recombination event.

[0048] In one embodiment, the nucleic acid or portion thereof encodes aprotein or polypeptide having at least one function characteristic of amammalian CXCR3 protein (e.g., a human CXCR3 receptor), such as abinding activity (e.g., ligand, inhibitor and/or promoter binding), asignalling activity (e.g., activation of a mammalian G protein,induction of rapid and transient increase in the concentration ofcytosolic free calcium [Ca²⁺]_(i)), and/or stimulation of a cellularresponse (e.g., stimulation of chemotaxis, exocytosis or inflammatorymediator release by leukocytes). The present invention also relates morespecifically to isolated and/or recombinant nucleic acids or a portionthereof comprising sequences which encode a mammalian CXCR3 receptor ora portion thereof. The present invention relates even more specificallyto isolated and/or recombinant nucleic acids comprising sequences whichencode a human CXCR3 protein.

[0049] The invention further relates to isolated and/or recombinantnucleic acids, including double or single stranded DNA or RNA, that arecharacterized by (1) their ability to hybridize to: (a) a nucleic acidhaving the sequence SEQ ID NO:1, (b) a nucleic acid having a sequencewhich is complementary to SEQ ID NO:1, or (c) a portion of the foregoingcomprising the open reading frame of SEQ ID NO:1 (a portion of thestrand illustrated in FIG. 1 or the corresponding portion of thecomplementary strand); and/or (2) by their ability to encode apolypeptide having the amino acid sequence SEQ ID NO:2 or a functionalequivalent thereof (i.e., a polypeptide having ligand binding activityfor one or more natural or physiological ligand(s) of the receptorand/or stimulatory function responsive to ligand binding, such that itcan induce a cellular response (e.g., induction (including triggering orstimulation) of chemotaxis, exocytosis or inflammatory mediator releaseby leukocytes); and/or (3) by both characteristics.

[0050] In one embodiment, the percent amino acid sequence identitybetween SEQ ID NO:2 and functional equivalents thereof is at least about60% (≧60%). In a preferred embodiment, functional equivalents of SEQ IDNO:2 share at least about 700 sequence identity with SEQ ID NOS:2. Morepreferably, the percent amino acid sequence identity between SEQ ID NO:2and functional equivalents thereof is at least about 80%, and still morepreferably, at least about 90%.

[0051] Isolated and/or recombinant nucleic acids meeting these criteriacomprise nucleic acids having sequences identical to sequences ofnaturally occurring mammalian CXCR3 receptors and portions thereof, orvariants of the naturally occurring sequences. Such variants includemutants differing by the addition, deletion or substitution of one ormore residues, modified nucleic acids in which one or more residues ismodified (e.g., DNA or RNA analogs), and mutants comprising one or moremodified residues. In one embodiment, the nucleic acid shares at leastabout 50% nucleotide sequence similarity, more preferably at least about75% nucleotide sequence similarity, and still more preferably at leastabout 90% nucleotide sequence similarity, with one strand of thesequence illustrated in SEQ ID NO:1 or to the coding region thereof.Preferred nucleic acids have lengths of at least about 40 nucleotides,more preferably at least about 50, and still more preferably at leastabout 75 nucleotides.

[0052] Such nucleic acids can be detected and isolated by hybridizationunder high stringency conditions or moderate stringency conditions, forexample. “High stringency conditions” and “moderate stringencyconditions” for nucleic acid hybridizations are explained on pages2-10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in CurrentProtocols in Molecular Biology (Ausubel, F. M et al., eds., Vol. 1,Suppl. 26, 1991), the teachings of which are incorporated herein byreference. Factors such as probe length, base composition, percentmismatch between the hybridizing sequences, temperature and ionicstrength influence the stability of nucleic acid hybrids. This, high ormoderate stringency conditions can be determined empirically to achievethe desired selectivity.

[0053] Isolated and/or recombinant nucleic acids that are characterizedby their ability to hybridize to a nucleic acid having the sequence SEQID NO:1 or the complement thereof (e.g., under high or moderatestringency conditions) may further encode a protein or polypeptidehaving at least one function characteristic of a mammalian CXCR3 protein(e.g., a human CXCR3 protein), such as a binding activity (e.g., ligand,inhibitor and/or promoter binding), a signalling activity (e.g.,activation of a mammalian G protein, induction of rapid and transientincrease in the concentration of cytosolic free calcium [Ca²⁺]_(i)),and/or stimulation of a cellular response (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes).

[0054] The human CXCR3 nucleic acid described herein, or sufficientportions thereof, whether isolated, recombinant and/or synthetic,including fragments produced by PCR, can be used as probes or primers todetect and/or recover nucleic acids (e.g., genomic DNA, allelicvariants, cDNA) encoding CXCR3 receptors (homologs) or other relatedreceptor genes (e.g., novel CXC chemokine receptor genes) from othermammalian species including, but not limited to primates (e.g., aprimate other than a human, such as a monkey (e.g., cynomolgus monkey)),bovine, ovine, equine, canine, feline and rodent (e.g., guinea pig,murine species such as rat, mouse). This can be achieved using theprocedures described herein or other suitable methods, includinghybridization, PCR or other suitable techniques. Mammalian nucleic acidscan be used to prepare constricts (e.g., vectors), receptor or fragmentsthereof, and host strains useful in the production and methods of use ofreceptor.

[0055] In one embodiment, a nucleic acid encoding a mammalian CXCR3protein (or variant) is producible by methods such as PCR amplification.For example, appropriate primers (e.g., a pair of primers or nestedprimers) can be designed which comprise a sequence which iscomplementary or substantially complementary to a portion of the humanCXCR3 cDNA described herein. For instance, primers complementary to the5′- or 3′-ends of the coding sequence and/or flanking the codingsequence can be designed. Such primers can be used in a polymerase chainreaction with a suitable template nucleic acid to obtain nucleic acidencoding a mammalian CXCR3, for example. Suitable templates includee.g., constructs described herein (such as pcDNA3-Clone8), a cDNA orgenomic library or another suitable source of mammalian (e.g., a human,primate) cDNA or genomic DNA. Primers can contain portions complementaryto flanking sequences of a construct selected as template asappropriate.

[0056] The binding function of a protein or polypeptide (e.g., encodedby hybridizing nucleic acid) can be detected in binding or bindinginhibition assays, using membrane fractions containing receptor or cellsexpressing receptor, for example (see e.g., Van Riper et al., J. Exp.Med., 177: 851-856 (1993); Sledziewski et al., U.S. Pat. No. 5,284,746(Feb. 8, 1994)). Thus, the ability of the encoded protein or polypeptideto bind a ligand, such as IP-10 or Mig, an inhibitor and/or promoter,can be assessed. The antigenic properties of proteins or polypeptidesencoded by nucleic acids of the present invention can be determined byimmunological methods employing antibodies that bind to a mammalianCXCR3, such as immunoblotting, immunoprecipitation and immunoassay(e.g., radioimmunoassay, ELISA).

[0057] The signalling function of a protein or polypeptide (e.g.,encoded by hybridizing nucleic acid) can be detected by enzymatic assaysfor G protein activity responsive to receptor binding (e.g., exchange ofGTP for GDP on the G protein a subunit, using membrane fractions). Gprotein coupling can be further assessed, for example, using assays inwhich stimulation by G protein is blocked by treatment or pre-treatmentof cells or a suitable cellular fraction (e.g., membranes) with specificinhibitors of G proteins, such as Bordetella pertussis toxin (Bischoff,S. C. et al., Eur. J. Immunol., 23: 761-767 (1993); Sozzani, S. et al.,J. Immunol., 147: 2215-2221 (1991)).

[0058] The stimulatory function of a protein or polypeptide (e.g.,encoded by hybridizing nucleic acid) can be detected by standard assaysfor chemotaxis or mediator release, using cells expressing the proteinor polypeptide (e g., assays which monitor chemotaxis, exocytosis (e.g.,degranulation of enzymes, such as esterases (e.g., serine esterases),perforin, granzymes) or mediator release (e.g., histamine, leukotriene)in response to a ligand (e.g., a chemokine such as IP-10 or Mig) or apromoter (see e.g., Taub, D. D. et al., J. Immunol., 155: 3877-3888(1995); Baggliolini, M. and C. A. Dahinden, Immunology Today, 15:127-133 (1994) and references cited therein). Functions characteristicof a mammalian CXCR3 receptor may also be assessed by other suitablemethods.

[0059] These methods, alone or in combination with other suitablemethods can also be used in procedures for the identification and/orisolation of nucleic acids which encode a polypeptide having the aminoacid sequence SEQ ID NO:2 or functional equivalents thereof, and havingan activity detected by the assay. Portions of isolated nucleic acidswhich encode polypeptide portions of SEQ ID NO:2 having a certainfunction can be also identified and isolated in this manner.

[0060] Nucleic acids of the present invention can be used in theproduction of proteins or polypeptides. For example, a nucleic acidcontaining all or part of the coding sequence for a mammalian CXCR3receptor, or DNA which hybridizes to the sequence SEQ ID NO:1, or thecomplement thereof, can be incorporated into a construct for furthermanipulation of sequences or for production of the encoded polypeptidein suitable host cells. Nucleic acids of the present invention can alsobe modified, for example, by incorporation of or attachment (directly orindirectly) of a detectable label such as a radioisotope, spin label,antigen or enzyme label, flourescent or chemiluminesent group and thelike, and such modified forms are included within the scope of theinvention.

[0061] Antisense Constructs

[0062] In another embodiment, the nucleic acid is an antisense nucleicacid, which is complementary, in whole or in part, to a target moleculecomprising a sense strand, and can hybridize with the target molecule.The target can be DNA, or its RNA counterpart (i.e., wherein T residuesof the DNA are U residues in the RNA counterpart). When introduced intoa cell using suitable methods, antisense nucleic acid can inhibit theexpression of the gene encoded by the sense strand. Antisense nucleicacids can be produced by standard techniques.

[0063] In one embodiment, the antisense nucleic acid is wholly orpartially complementary to and can hybridize with a target nucleic acid,wherein the target nucleic acid can hybridize to a nucleic acid havingthe sequence of the complement of SEQ ID NO:1. For example, antisensenucleic acid can be complementary to a target nucleic acid having thesequence of SEQ ID NO:1 or a portion thereof sufficient to allowhybridization. In another embodiment, the antisense nucleic acid iswholly or partially complementary to and can hybridize with a targetnucleic acid which encodes a mammalian CXCR3 receptor (e.g., humanIP-10/Mig receptor CXCR3).

[0064] Antisense nucleic acids are useful for a variety of purposes,including research and therapeutic applications. For example, aconstruct comprising an antisense nucleic acid can be introduced into asuitable cell to inhibit receptor expression. Such a cell provides avaluable control cell, for instance in assessing the specificity ofreceptor-ligand interaction with the parent cell or other related celltypes. In another aspect, such a construct can be introduced into someor all of the cells of a mammal. The antisense nucleic acid inhibitsreceptor expression, and inflammatory processes mediated by CXCR3receptors in the cells containing the construct can be inhibited. Thus,an inflammatory disease or condition can be treated using an antisensenucleic acid of the present invention. Suitable laboratory animalscomprising an antisense construct can also provide useful models fordeficiencies of leukocyte function, and of activated T lymphocytedeficiency in particular, and can provide further information regardingCXCR3 receptor function. Such animals can provide valuable models ofinfectious disease or cancer, useful for elucidating the role ofleukocytes, such as T lymphocytes and NK cells, in host defenses.

[0065] Method of Producing Recombinant Proteins

[0066] Another aspect of the invention relates to a method of producinga mammalian CXCR3 protein or variant (e.g., portion) thereof.Recombinant protein can be obtained, for example, by the expression of arecombinant nucleic acid (e.g., DNA) molecule encoding a mammalian CXCR3or variant thereof in a suitable host cell, for example.

[0067] Constructs (e.g., expression vectors) suitable for the expressionof a mammalian CXCR3 protein or variant thereof are also provided. Theconstructs can be introduced into a suitable host cell, and cells whichexpress a recombinant mammalian CXCR3 protein or variant thereof can beproduced and maintained in culture. Such cells are useful for a varietyof purposes, including use in the production of protein forcharacterization, isolation and/or purification, (e.g., affinitypurification), use as immunogen, and in binding assays or otherfunctional assays (e.g., to screen for ligands, inhibitors and/orpromoters of receptor function), for instance. Suitable host cells canbe procaryotic, including bacterial cells such as E. coli, B. subtilisand or other suitable bacteria, or eucaryotic, such as fungal or yeastcells (e.g., Pichia pastoris, Aspergilius species, Saccharomzycescerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or otherlower eucaryotic cells, and cells of higher eucaryotes such as thosefrom insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor, publishedNov. 24, 1994)) or mammals (e.g., Chinese hamster ovary cells (CHO), COScells, HuT 78 cells, 293 cells). (See, e.g., Ausubel, F. M. et al., eds.Current Protocols in Molecular Biology, Greene Publishing Associates andJohn Wiley & Sons Inc., (1993)).

[0068] Host cells which produce a recombinant mammalian CXCR3 protein orvariant thereof can be produced as follows. For example, a nucleic acidencoding all or part of the coding sequence for the desired protein canbe inserted into a nucleic acid vector, e.g., a DNA vector, such as aplasmid, virus or other suitable replicon for expression. A variety ofvectors are available, including vectors which are maintained in singlecopy or multiple copy, or which become integrated into the host cellchromosome.

[0069] Transcriptional and/or translational signals of a mammalian CXCR3gene can be used to direct expression. Suitable expression vectors forthe expression of a nucleic acid encoding all or part of the codingsequence of the desired protein are also available. Suitable expressionvectors can contain a number of components, including, but not limitedto one or more of the following: an origin of replication; a selectablemarker gene; one or more expression control elements, such as atranscriptional control element (e.g., a promoter, an enhancer,terminator), and/or one or more translation signals; a signal sequenceor leader sequence for membrane targeting in a selected host cell (e.g.,of mammalian origin or from a heterologous mammalian or non-mammalianspecies). In a construct, a signal sequence can be provided by thevector, the mammalian CXCR3 coding sequence, or other source. Sequencespresent at a site of integration can also provide these elements.

[0070] A promoter can be provided for expression in a suitable hostcell. Promoters can be constitutive or inducible. For example, apromoter can be operably linked to a nucleic acid encoding the mammalianCXCR3 protein or variant thereof, such that it directs expression of theencoded polypeptide. A variety of suitable promoters for procaryotic(e.g., lac, tac, T3, T7 promoters for E. coli) and eucaryotic (e.g.,yeast alcohol dehydrogenase (ADH1), SV40, CMV) hosts are available.

[0071] In addition, the expression vectors typically comprise aselectable marker for selection of host cells carrying the vector, and,in the case of replicable expression vector, an origin or replication.Genes encoding products which confer antibiotic or drug resistance arecommon selectable markers and may be used in procaryotic (e.g.,β-lactamase gene (ampicillin resistance), Tet gene for tetracyclineresistance) and eucaryotic cells (e.g., neomycin (G418 or geneticin),gpt (mycophenolic acid), ampicillin, or hygromycin resistance genes).Dihydrofolate reductase marker genes permit selection with methotrexatein a variety of hosts. Genes encoding the gene product of auxotrophicmarkers of the host (e.g., LEU2, URA3, HIS3) are often used asselectable markers in yeast. Use of viral (e.g., baculovirus) or phagevectors, and vectors which are capable of integrating into the genome ofthe host cell, such as retroviral vectors, are also contemplated. Thepresent invention also relates to cells carrying these expressionvectors.

[0072] For example, a nucleic acid encoding a mammalian CXCR3 protein orvariant thereof, or a construct comprising such nucleic acid, can beintroduced into a suitable host cell by a method appropriate to the hostcell selected (e.g., transformation, transfection, electroporation,infection) such that the nucleic acid is operably linked to one or moreexpression control elements (e.g., in a vector, in a construct createdby processes in the cell, integrated into the host cell genome). Hostcells can be maintained under conditions suitable for expression (e.g.,in the presence of inducer, suitable media supplemented with appropriatesalts, growth factors, antibiotic, nutritional supplements, etc.),whereby the encoded polypeptide is produced. If desired, the encodedprotein (e.g., human CXCR3) can be isolated (e.g., from the host cells,medium, milk). It will be appreciated that the method encompassesexpression in a host cell of a transgenic animal (see e.g., WO 92/03918,GenPharm International, published Mar. 19, 1992).

[0073] Fusion proteins can also be produced in this manner. For example,some embodiments can be produced by the insertion of a mammalian CXCR3protein cDNA or portion thereof into a suitable expression vector, suchas Bluescript®II SK+/− (Stratagene), pGEX-4T-2 (Pharmacia), pcDNA-3(Invitrogen) or pET-15b (Novagen). The resulting construct can beintroduced into a suitable host cell for expression. Upon expression,fusion protein can be isolated or purified from a cell lysate by meansof a suitable affinity matrix (see e.g., Current Protocols in MolecularBiology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26, pp.16.4.1-16.7.8 (1991)). In addition, affinity labels provide a means ofdetecting a fusion protein. For example, the cell surface expression orpresence in a particular cell fraction of a fusion protein comprising anantigen or epitope affinity label can be detected by means of anappropriate antibody.

[0074] Antibodies

[0075] The invention further relates to antibodies reactive with amammalian CXCR3 protein or portion thereof. In a preferred embodiment,the antibodies specifically bind mammalian CXCR3 receptor(s) or aportion thereof. In one embodiment, antibodies are raised against anisolated and/or recombinant mammalian CXCR3 protein or portion thereof(e.g., a peptide) or against a host cell which expresses recombinantmammalian CXCR3.

[0076] Antibodies which can inhibit one or more functions characteristicof a mammalian (e.g., a primate such as a human) CXCR3 protein, such asa binding activity, a signalling activity, and/or stimulation of acellular response, are also encompassed by the present invention. In oneembodiment, antibodies of the present invention can inhibit binding of aligand (i.e., one or more ligands) to a mammalian CXCR3 protein and/orcan inhibit one or more functions mediated by a mammalian CXCR3 proteinin response to ligand binding. In a particularly preferred embodiment,the antibodies can inhibit (reduce or prevent) the interaction ofreceptor with a natural ligand, such as IP-10 and/or Mig. For example,as shown herein, antibodies of the present invention can selectivelyinhibit the interaction of a human CXCR3 protein with IP-10 and/orselectively inhibit receptor functions in response thereto (e.g.,signalling activity and/or a cellular response). An antibody designated1CG, which displays this selectivity, can inhibit binding of IP-10 to ahuman CXCR3 protein as well as calcium flux and chemotaxis induced byIP-10, but does not significantly inhibit calcium flux induced by Migunder the same conditions. As shown herein, additional antibodies caninhibit IP-10 binding to CXCR3 (e.g., 3A8, 2F8, 3A12, 3E2, 4B4, 4D2,5B12, 7B8 and 8D5) or IP-10-induced chemotaxis (e.g., 1A5, 3A8, 5F10,and 10C6), although these antibodies did not inhibit Mig-inducedsignalling under the conditions used, indicating that they are alsoselective inhibitors of the interaction of a human CXCR3 protein withIP-10 and/or of receptor functions in response thereto.

[0077] In a particularly preferred embodiment, the antibodies of thepresent invention have specificity for a human CXCR3 protein, and evenmore preferably have an epitopic specificity which is the same as orsimilar to that of a murine monoclonal antibody (mAb) designated 1C6.Antibodies having an epitopic specificity which is the same as orsimilar to that of mAb 1C6 can be identified using one or more suitabletechniques for characterizing epitopic specificity. For example,antibodies having an epitopic specificity which is the same as orsimilar to that of mAb 1C6 can be identified by their ability to competewith murine mAb 1C6 for binding to a human CXCR3 protein or portionthereof (e.g., to cells bearing human CXCR3, including lymphocytes suchas activated T cells, NK cells, or recombinant host cells comprising anucleic acid of the present invention). In one embodiment, antibodieshaving an epitopic specificity which is the same as or similar to thatof mAb 1C6 are further characterized by the ability of a polypeptidehaving a sequence which is the same as that of residues 1-15 (“P1”) ofSEQ ID NO:2 to inhibit binding of the antibodies to a human CXCR3protein in a suitable assay (see e.g., Example 8). In one aspect of thisembodiment, binding to a human CXCR3 protein by such antibodies is notsignificantly inhibited by a polypeptide having a sequence which is thesame as that of residues 16-30 (“P2”) or 31-45 (“P3”) of SEQ ID NO:2.

[0078] Other antibodies encompassed by the present invention includeantibodies which can bind to a human CXCR3 protein, wherein said bindingcan be inhibited by a portion of SEQ ID NO:2 corresponding to theN-terminal extracellular segment or a portion thereof. Suitable portionsof the N-terminal extracellular segment include N-terminal, internal orC-terminal portions of that segment, such as a polypeptide comprisingthe N-terminus and having a sequence which is the same as that ofresidues 1-30, 1-45 or 1-58 of SEQ ID NO:2, for example, or apolypeptide having a sequence which is the same as that of residues16-30 or 45-58 of SEQ ID NO:2. In a preferred embodiment, such portionshave “at least one immunological property” of a mammalian CXCR3 proteinas defined hereinabove, wherein the mammal is a human. For example,antibodies reactive with a human CXCR3 protein for which binding can beinhibited by a polypeptide having a sequence which is the same as thatof residues 16-30 of SEQ ID NO:2 have been obtained (Example 9).

[0079] The antibodies of the present invention can be polyclonal ormonoclonal, and the term antibody is intended to encompass bothpolyclonal and monoclonal antibodies. The terms polyclonal andmonoclonal refer to the degree of homogeneity of an antibodypreparation, and are not intended to be limited to particular methods ofproduction.

[0080] Antibodies of the present invention can be raised against anappropriate immunogen, including proteins or polypeptides of the presentinvention, such as isolated and/or recombinant mammalian CXCR3 proteinor portion thereof (including synthetic molecules such as syntheticpeptides). In addition, cells expressing recombinant mammalian CXCR3,such as transfected cells, can be used as immunogens or in a screen forantibody which binds receptor. See for example, Chuntharapai et al., J.Immunol., 152: 1783-1789 (1994); and Chuntharapai et al., U.S. Pat. No.5,440,021.

[0081] Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed using any suitable technique. Avariety of methods have been described (see e.g., Kohler et al., Nature,256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.),Chapter 11, (1991)). Generally, a hybridoma can be produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as SP2/0)with antibody producing cells. The antibody producing cell, preferablythose of the spleen or lymph nodes, can be obtained from animalsimmunized with the antigen of interest. The fused cells (hybridomas) canbe isolated using selective culture conditions, and cloned by limitingdilution. Cells which produce antibodies with the desired specificitycan be selected by a suitable assay (e.g., ELISA).

[0082] Other suitable methods of producing or isolating antibodies ofthe requisite specificity can used, including, for example, methodswhich select recombinant antibody from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies (see e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Lonberg et al., U.S. Pat. No. 5,545,1806; Surani etal., U.S. Pat. No. 5,545,807).

[0083] Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted), or veneered antibodies, as well as chimeric, CDR-graftedor veneered single chain antibodies, comprising portions derived fromdifferent species, and the like are also encompassed by the presentinvention and the term “antibody”. The various portions Or theseantibodies can be joined together chemically by conventional techniques,or a polypeptide(s) can be prepared as a contiguous protein usinggenetic engineering techniques. The term “humanized antibody orimmunoglobulin” as used herein refers to an antibody or immunoglobulincomprising portions of immunoglobulins of different origin, wherein atleast one portion is of human origin. Accordingly, the present inventionrelates to a humanized antibody (including antigen-binding fragmentsthereof) having binding specificity for a mammalian CXCR3 protein (e.g.,a human CXCR3 protein), comprising antigen binding regions of nonhumanorigin (e.g., rodent) and at least of portion of an immunoglobulin ofhuman origin (e.g., a human framework region, a human constant region,or portions thereof). For example, humanized antibodies can compriseportions derived from an immunoglobulin of nonhuman origin with therequisite specificity (e.g., a nonhuman mouse variable region) and fromimmunoglobulin sequences of human origin (e.g., a human constant regionor portion thereof) joined together chemically or prepared as acontiguous polupeptide using genetic engineering techniques. Anotherexample of a humanized antibody of the invention is an immunoglobulincomprising one or more immunoglobulin chains, said immunoglobulincomprising a CDR of nonhuman origin (e.g., one or more CDRs derived froman antibody of nonhuman origin) and a framework region derived from alight and/or heavy chain of human origin (e.g., CDR-grafted antibodieswith or without framework changes). In one aspect of this embodiment,the immunoglobulin comprises the light chain CDRs (CDR1, CDR2 and CDR3)and heavy chain CDRs (CDR1, CDR2, CDR3) of a nonhuman immunoglobulin. Inone embodiment, the humanized immunoglobulin has an epitopic specificitywhich is the same or similar to that mAB 1C6. In a preferred embodiment,the antigen binding region of the humanized immunoglobulin is derivedfrom mAb 1C6. The term humanized antibody also encompasses single chainantibodies. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabillyet al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No.4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M.S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No.0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European PatentNo. 0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; andPadlan, E. A. et al., EP 0 519 596 A1. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, andLadner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,242: 423-426 (1988)) regarding single chain antibodies.

[0084] In addition, functional fragments of antibodies, includingfragments of chimeric, humanized, primatized, veneered or single chainantibodies, can also be produced. Functional fragments of foregoingantibodies retain at least one binding function and/or modulationfunction of the full-length antibody from which they are derived. Forexample, antibody fragments capable of binding to a mammalian CXCR3protein or portion thereof, including, but not limited to, Fv, Fab, Fab′and F(ab′)₂ fragments are encompassed by the invention. Such fragmentscan be produced by enzymatic cleavage or by recombinant techniques. Forinstance, papain or pepsin cleavage can generate Fab or F(ab′)₂fragments, respectively. Antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain portion can be designed toinclude DNA sequences encoding the CH₁ domain and hinge region of theheavy chain.

[0085] The antibodies of the present invention are useful in a varietyof applications, including research, diagnostic and therapeuticapplications. For instance, they can be used to isolate, purify and/ordetect receptor or portions thereof, and to study receptor structure(e.g., conformation) and function.

[0086] The antibodies of the present invention can also be used tomodulate receptor function in research and therapeutic applications. Forinstance, antibodies can act as inhibitors to inhibit (reduce orprevent) (a) binding (e.g., of a ligand, a second inhibitor or apromoter) to the receptor, (b) receptor signalling, and/or (c) acellular response. Antibodies which act as inhibitors of receptorfunction can block ligand or promoter binding directly or indirectly(e.g., by causing a conformational change in the receptor). For example,antibodies can inhibit receptor function by inhibiting binding of aligand, or by desensitization (with or without inhibition of binding ofa ligand). Antibodies of the present invention can be used asantagonists of effector cells such as activated or stimulated Tlymphocytes and natural killer (NK) cells, and find use in methods oftherapy (including prophylaxis) of diseases or conditions which can betreated with inhibitors of CXCR3 function as described herein.Antibodies which can selectively inhibit the interaction of a humanCXCR3 protein with IP-10 and/or selectively inhibit receptor functionsin response thereto (e.g., mAb 1C6, antibodies having an epitopicspecificity similar to that of mAb 1C6) are particularly useful in thetreatment of diseases or disorders mediated by IP-10-CXCR3 interaction.For example, inflammatory conditions such as psoriasis, inflammatorybowel diseases, nephritis, and multiple sclerosis can be particularlyamenable to therapy.

[0087] Surprisingly, mAb 1C6 inhibits T cell activation as assessed in amixed lymphocyte reaction (MLR). Accordingly, mAb 1C6 and otheranti-CXCR3 antibodies which can inhibit T cell activation, can be usedto inhibit T cell activation and function(s) associated with activation,such as cytokine production, cytotoxic T cell killing, and/or provisionof T cell help. When used in methods of therapy or prophylaxis asdescribed herein, such antibodies can have the added advantage ofinhibiting further T cell activation. Such antibodies are particularlyattractive as therapeutic agents for treating graft rejection (e.g., intransplantation), including allograft rejection or graft-versus-hostdisease, or other diseases or conditions in which inhibition of theactivation of T cells is desired.

[0088] Antibodies which bind receptor can also act as agonists ofreceptor function, triggering or stimulating a receptor function, suchas signalling and/or a cellular response (e.g., calcium flux,chemotaxis, exocytosis or pro-inflammatory mediator release) uponbinding to receptor. Accordingly, such antibodies can be used asagonists of effector cells such as activated or stimulated T lymphocytesand natural killer (NK) cells, and find use in methods of therapy(including prophylaxis) of diseases or conditions which can be treatedwith promoters of CXCR3 function as described herein.

[0089] In addition, the various antibodies of the present invention canbe used to detect or measure the expression of receptor, for example, onleukocytes such as activated T cells or natural killer cells (NK cells),or on cells transfected with a receptor gene. Thus, they also haveutility in applications such as cell sort.-ng (e.g., flow cytometry,fluorescence activated cell sorting), for diagnostic or researchpurposes.

[0090] Anti-idiotypic antibodies are also provided. Anti-idiotypicantibodies recognize antigenic determinants associated with theantigen-binding site of another antibody. Anti-idiotypic antibodies canbe prepared a against a first antibody by immunizing an animal of thesame species, and preferably of the same strain as the animal used toproduce the first antibody, with said first antibody. See e.g., U.S.Pat. No. 4,699,880.

[0091] In one embodiment, antibodies are raised against receptor or aportion thereof, and these antibodies are used in turn as immunogen toproduce an anti-idiotypic antibody. The anti-Id produced thereby canmimic receptor and bind compounds which bind receptor, such as ligands,inhibitors or promoters of receptor function, and can be used in animmunoassay to detect, identify or quantitate such compounds. Such ananti-idiotypic antibody can also be an inhibitor of receptor function,although it does not bind receptor itself.

[0092] Anti-idiotypic (i.e., Anti-Id) antibody can itself be used toraise an anti-idiotypic antibody (i.e., Anti-anti-Id). Such an antibodycan be similar or identical in specificity to the original immunizingantibody. In one embodiment, antibody antagonists which block binding toreceptor can be used to raise Anti-Id, and the Anti-Id can be used toraise Anti-anti-Id, which can have a specificity which is similar oridentical to that of the antibody antagonist. These anti-anti-Idantibodies can be assessed for inhibitory effect on receptor function todetermine if they are antagonists.

[0093] Single chain, and chimeric, humanized, primatized (CDR-grafted),veneered, as well as chimeric, CDR-grafted, or veneered single chainanti-idiotypic antibodies can be prepared, and are encompassed by theterm anti-idiotypic antibody. Antibody fragments of such antibodies canalso be prepared.

[0094] The antibodies and fragments of the present invention can bemodified, for example, by incorporation of or attachment (directly orindirectly) of a detectable label such as a radioisotope, spin label,antigen or enzyme label, flourescent or chemiluminesent group and thelike, and such modified forms are included within the scope of theinvention.

[0095] Identification of Ligands, Inhibitors or Promoters of ReceptorFunction

[0096] As used herein, a ligand is a substance which binds to a receptorprotein. A ligand of a selected mammalian CXCR3 protein is a substancewhich binds to the selected mammalian CXCR3 protein. In a preferredembodiment, ligand binding of a mammalian CXCR3 protein occurs with highaffinity. The term ligand refers to substances including, but notlimited to, a natural ligand, whether isolated and/or purified,synthetic, and/or recombinant, a homolog of a natural ligand (e.g., fromanother mammal), antibodies, portions of such molecules, and othersubstances which bind receptor. A natural ligand of a selected mammalianreceptor can bind to the receptor under physiological conditions, and isof a mammalian origin which is the same as that of the mammalian CXCR3protein. The term ligand encompasses substances which are inhibitors orpromoters of receptor activity, as well as substances which selectivelybind receptor, but lack inhibitor or promoter activity.

[0097] As used herein, an inhibitor is a substance which inhibits atleast one function characteristic of a mammalian CXCR3 protein (e.g., ahuman CXCR3), such as a binding activity (e.g., ligand binding, promoterbinding), a signalling activity (e.g., activation of a mammalian Gprotein, induction of rapid and transient increase in the concentrationof cytosolic free calcium [Ca²⁺]_(i)), and/or cellular response function(e.g., stimulation of chemotaxis, exocytosis or inflammatory mediatorrelease by leukocytes). The term inhibitor refers to substancesincluding antagonists which bind receptor (e.g., an antibody, a mutantof a natural ligand, other competitive inhibitors of ligand binding),and substances which inhibit receptor function without binding thereto(e.g., an anti-idiotypic antibody).

[0098] As used herein, a promoter is a substance which promotes (inducesor enhances) at least one function characteristic of a mammalian CXCR3protein (e.g., a human CXCR3), such as a binding activity (e.g., ligand,inhibitor and/or promoter binding), a signalling activity (e.g.,activation of a mammalian G protein, induction of rapid and transientincrease in the concentration of cytosolic free calcium [Ca²⁺]_(i)),and/or a cellular response function (e.g., stimulation of chemotaxis,exocytosis or inflammatory mediator release by leukocytes). The termpromoter refers to substances including agonists which bind receptor(e.g., an antibody, a homolog of a natural ligand from another species),and substances which promote receptor function without binding thereto(e.g., by activating an associated protein). In a preferred embodiment,the agonist is other than a homolog of a natural ligand.

[0099] The assays described below, which rely upon the nucleic acids andproteins of the present invention, can be used, alone or in combinationwith each other or other suitable methods, % identify ligands,inhibitors or promoters of a mammalian CXCR3 protein or variant. The invitro methods of the present invention can be adapted forhigh-throughput screening in which large numbers of samples areprocessed (e.g., a 96 well format). Host cells comprising a nucleic acidof the present invention and expressing recombinant mammalian CXCR3(e.g., human CXCR3) at levels suitable for high-throughput screening canbe used, and thus, are particularly valuable in the identificationand/or isolation of ligands, inhibitors and promoters of mammalian CXCR3proteins. Expression of receptor can be monitored in a variety of ways.For instance, expression can be monitored using antibodies of thepresent invention which bind receptor or a portion thereof. Also,commercially available antibodies can be used to detect expression of anantigen- or epitope-tagged fusion protein comprising a receptor proteinor polypeptide (e.g., FLAG tagged receptors), and cells expressing thedesired level can be selected.

[0100] Nucleic acid encoding a mammalian CXCR3 protein, the can beincorporated into an expression system to produce a receptor protein orpolypeptide as described above. An isolated and/or recombinant receptorprotein or polypeptide, such as a receptor expressed in cells stably ortransiently transfected with a construct comprising a nucleic acid ofthe present invention, or in a cell fraction containing receptor (e.g.,a membrane fraction from transfected cells, liposomes incorporatingreceptor) can be used in tests for receptor function. The receptor canbe further purified if desired. Testing of receptor function can becarried out in vitro or in vivo.

[0101] An isolated and/or recombinant mammalian CXCR3 protein, such as ahuman CXCR3 as shown in FIG. 2 (SEQ ID NO:2), can be used in the presentmethod, in which the effect of a compound is assessed by monitoringreceptor function as described herein or using other suitabletechniques. For example, stable or transient transfectants such as thosedescribed in Example 2 or other suitable cells (e.g., baculovirusinfected Sf9 cells, stable tranfectants of mouse L1-2 pre-B cells(derived from a pre-B lymphoma, Dr. Eugene Butcher (Stanford University,Stanford, Calif.)), can be used in binding assays. Stable transfectantsof Jurkat cells (Example 2) or of other suitable cells capable ofchemotaxis can be used (e.g., mouse L1-2 pre-B cells) in chemotaxisassays, for example.

[0102] According to the method of the present invention, compounds canbe individually screened or one or more compounds can be testedsimultaneously according to the methods herein. Where a mixture ofcompounds is tested, the compounds selected by the processes describedcan be separated (as appropriate) and identified by suitable methods(e.g., PCR, sequencing, chromatography). The presence of one or morecompounds (e.g., a ligand, inhibitor, promoter) in a test sample canalso be determined according to these methods.

[0103] Large combinatorial libraries of compounds (e.g., organiccompounds, recombinant or synthetic peptides, “peptoids”, nucleic acids)produced by combinatorial chemical synthesis or other methods can betested (see e.g., Zuckerman, R. N. et al., J. Med. Chem., 37: 267B-2685(1994) and references cited therein; see also, Ohlmeyer, M. H. J. etal., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H.et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating totagged compounds; Ratter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner,V. D. et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. PatentNo. 4,833,092). Where compounds selected from a combinatorial library bythe present method carry unique tags, identification of individualcompounds by chromatographic methods is possible.

[0104] In one embodiment, phage display methodology is used. Forexample, receptor can be contacted with a phage (e.g., a phage orcollection of phage such as a library) displaying a polypeptide underconditions appropriate for receptor binding (e.g., in a suitable bindingbuffer). Phage bound to receptor can be selected using standardtechniques or other suitable methods. Phage can be separated fromreceptor using a suitable elution buffer. For example, a change in theionic strength or pH card lead to a release of phage. Alternatively, theelution buffer can comprise a release component or components designedto disrupt binding of compounds (e.g., one or more compounds which candisrupt binding of the displayed peptide to the receptor, such as aligand, inhibitor, and/or promoter which competitively inhibitsbinding). Optionally, the selection process can be repeated or anotherselection step can be used to further enrich for phage which bindreceptor. The displayed polypeptide can be characterized (e.g., bysequencing phage DNA). The polypeptides identified can be produced andfurther tested for ligand binding, inhibitor and/or promoter function.Analogs of such peptides can be produced which will have increasedstability or other desirable properties.

[0105] In one embodiment, phage expressing and displaying fusionproteins comprising a coat protein with an N-terminal peptide encoded byrandom sequence nucleic acids can be produced. Suitable host cellsexpressing a receptor protein or polypeptide of the present inventionare contacted with the phage, bound phage are selected, recovered andcharacterized. (See e.g., Doorbar, J. and G. Winter, J. Mol. Biol., 244:361 (1994) discussing a phage display procedure used with a Gprotein-coupled receptor).

[0106] Other sources of potential ligands, inhibitors and/or promotersof mammalian CXCR3 proteins include, but are not limited to, variants ofCXCR3 ligands, including naturally occurring, synthetic or recombinantvariants of IP-10 or Mig, substances such as other chemoattractants orchemokines, variants thereof, other inhibitors and/or promoters (e.g.,anti-CXCR3 antibodies, antagonists, agonists), other G-protein coupledreceptor ligands, inhibitors and/or promoters (e.g., antagonists oragonists), and soluble portions of a mammalian CXCR3 receptor, such as asuitable receptor peptide or analog which can inhibit receptor function(see e.g., Murphy, R. B., WO 94/05695).

[0107] Binding Assays

[0108] The isolated and/or recombinant receptor proteins or functionalvariants thereof, including portions thereof or suitable fusionproteins, can be used in a method to select and identify agents whichbind to a (one or more) mammalian CXCR3 protein, such as human CXCR3,and which are ligands, or potential inhibitors or promoters of receptoractivity. Agents selected by the method, including ligands, inhibitorsor promoters, can be further assessed for an inhibitory or stimulatoryeffect on receptor function and/or for therapeutic utility.

[0109] In one embodiment, an agent which binds to an active, isolatedand/or recombinant mammalian CXCR3 protein or polypeptide is identifiedby the method. In this embodiment, the receptor protein or polypeptideused has at least one property, activity or function characteristic of amammalian CXCR3 protein (as defined herein), such as a binding activity(e.g., ligand, inhibitor and/or promoter binding), a signalling activity(e.g., activation of a mammalian G protein, induction of rapid andtransient increase in the concentration of cytosolic free calcium[Ca²⁺]_(i)), cellular response function (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes),and/or an immunological property as defined herein. In a preferredembodiment, the isolated and/or recombinant mammalian CXCR3 protein orvariant has ligand binding function, and more preferably binds a naturalligand of the receptor. In a particularly preferred embodiment, theisolated and/or recombinant protein is a human CXCR3 protein encoded bythe nucleic acid illustrated FIG. 1 (SEQ ID NO:1).

[0110] For example, a composition comprising an isolated and/orrecombinant mammalian CXCR3 protein or variant thereof can be maintainedunder conditions suitable for binding, the receptor can be contactedwith an agent (e.g., a composition comprising one or more agent) to betested, and binding is detected or measured. In one embodiment, areceptor protein can be expressed in cells stably or transientlytransfected with a construct comprising a nucleic acid sequence whichencodes a receptor of the present invention. The cells can be maintainedunder conditions appropriate for expression of receptor. The cells arecontacted with an agent under conditions suitable for binding (e.g., ina suitable binding buffer), and binding can be detected by standardtechniques. For example, the extent of binding can be determinedrelative to a suitable control (e.g., compared with backgrounddetermined in the absence of agent, compared with binding of a secondagent (i.e., a standard), compared with binding of the agent tountransfected cells). Optionally, a cellular fraction, such as amembrane fraction, containing receptor can be used in lieu of wholecells.

[0111] Binding or complex formation can be detected directly orindirectly. In one embodiment, the agent can be labeled with a suitablelabel (e.g., fluorescent label, chemiluminescent label, isotope label,enzyme label), and binding can be determined by detection of the label.Specificity of binding can be assessed by competition or displacement,for example, using unlabeled agent or a ligand (e.g., IP-10, Mig) ascompetitor.

[0112] Ligands of the mammalian receptor, including natural ligands fromthe same mammalian species or from another species, can be identified inthis manner. The binding activity of a promoter or inhibitor which bindsreceptor can also be assessed using such a ligand binding assay.

[0113] Binding inhibition assays can also be used to identify ligands,and inhibitors and promoters which bind receptor and inhibit binding ofanother agent such as a ligand. For example, a binding assay can beconducted in which a reduction in the binding of a first agent (in theabsence of a second agent), as compared with binding of the first agentin the presence of the second test agent, is detected or measured. Thereceptor can be contacted with the first and second agentssimultaneously, or one after the other, in either order. A reduction inthe extent of binding of the first agent in the presence of the secondtest agent, is indicative of inhibition of binding by the second agent.For example, binding of the first agent could be decreased or abolished.

[0114] In one embodiment, direct inhibition of the binding of a firstagent (e.g., a chemokine such as IP-10, Mig) to a human CXCR3 by asecond test agent is monitored. For example, the ability of an agent toinhibit the binding of ¹²⁵I-labeled Mig to human CXCR3 can be monitored.Such an assay can be conducted using whole cells (e.g., a suitable cellline containing nucleic acid encoding a human CXCR3 receptor), or amembrane fraction from said cells, for instance.

[0115] Other methods of identifying the presence of an agent(s) whichbinds a receptor are available, such as methods which monitor eventswhich are triggered by receptor binding, including signalling functionand/or stimulation of a cellular response.

[0116] It will be understood that the inhibitory effect of antibodies ofthe present invention can be assessed in a binding inhibition assay.Competition between antibodies for receptor binding can also be assessedin the method in which the first agent in the assay is another antibody,under conditions suitable for antibody binding.

[0117] Ligands, receptor-binding inhibitors and promoters, which areidentified in this manner, can be further assessed to determine whether,subsequent to binding, they act to inhibit or activate other functionsof CXCR3 receptors and/or to assess their therapeutic utility.

[0118] Signalling Assays

[0119] The binding of a G protein-coupled receptor (e.g., by an agonist)can result in signalling by the receptor, and stimulation of theactivity of G protein. The induction of signalling function by an agentcan be monitored using any suitable method. For example, C proteinactivity, such as hydrolysis of GTP to GDP, or later signalling eventstriggered by receptor binding, such as induction of rapid and transientincrease in the concentration of intracellular (cytosolic) free calcium[Ca²⁺]_(i), can be assayed by methods known in the art or other suitablemethods (Example 2; see also, Neote, K. et al., Cell, 72: 415-425 1993);Van Riper et al., J. Exp. Med., 177: 851-856 (1993); Dahinden, C. A. etal., J. Exp. Med., 179: 751-75G (1994)).

[0120] The functional assay of Sledziewski et al. using hybrid G proteincoupled receptors can also be used to identify a ligand or promoter byits ability to activate a hybrid G protein or to identify an inhibitorby its ability to inhibit such activation (Sledziewski et al., U.S. Pat.No. 5,284,746, the teachings of which are incorporated herein byreference). In one embodiment, a biological response of the host cell(triggered by binding to hybrid receptor) can be monitored, detection ofthe response being indicative of the presence of ligand in the testsample. For example, a method of detecting the presence of a ligand in atest sample is described, wherein the ligand is an agent which iscapable of being bound by the ligand-binding domain of a receptor. Inone embodiment of the method, yeast host cells are transformed with aDNA construct capable of directing the expression of a biologicallyactive hybrid G protein-coupled receptor (i.e., a fusion protein). Thehybrid receptor comprises a mammalian G protein-coupled receptor havingat least one domain other than the ligand-binding domain replaced with acorresponding domain of a yeast G protein-coupled receptor, such as aSTE2 gene product. The yeast host cells containing the construct aremaintained under conditions in which the hybrid receptor is expressed,and the cells are contacted with a test sample under conditions suitableto permit binding of ligand to the hybrid receptor. A biologicalresponse of the host cell (triggered by binding to hybrid receptor) ismonitored, detection of the response being indicative of a signallingfunction. For instance, binding to a hybrid receptor derived from STE2gene product can lead to induction of the BAR1 promoter. Induction ofthe promoter can be measured by means of a reporter gene (e.g., β-gal),which is linked to the BAR1 promoter and introduced into host cells on asecond construct. Expression of the reporter gene can be detected by anin vitro enzyme assay on cell lysates or by the presence of bluecolonies on plates containing an indicator (e.g., X-gal) in the medium,for example.

[0121] In another embodiment, the assay can be used to identifypotential inhibitors of receptor function. The inhibitory activity of anagent can be determined using a ligand or promoter in the assay, andassessing the ability of the test agent to inhibit the activity inducedby ligand or promoter.

[0122] Variants of known ligands can also be screened for reducedability (decreased ability or no ability) to stimulate activity of acoupled G protein. In this embodiment, although the agent has ligandbinding activity (as determined by another method), engagement of thereceptor does not trigger or only weakly triggers activity of a coupledG protein. Such agents are potential antagonists, and can be furtherassessed for inhibitory activity.

[0123] Chemotaxis and Other Assays of Cellular Responses

[0124] Chemotaxis assays can also be used to assess receptor function.These assays are based on the functional migration of cells in vitro orin vivo induced by an agent, and can be used to assess the bindingand/or effect on chemotaxis of ligands, inhibitors, or promoters.

[0125] The use of an in vitro chemotaxis assay to assess the response ofcells to IP-10 and Mig is described in Example 2. Springer et al.describe a transendothelial lymphocyte chemotaxis assay (Springer etal., WO 94/20142, published Sep. 15, 1994, the teachings of which areincorporated herein by reference; see also Berman et al., ImmunolInvest., 17: 625-677 (1988)). Migration across endothelium into collagengels has also been described (Kavanaugh et al., J. Immunol, 146:4149-4156 (1991)).

[0126] Generally, chemotaxis assays monitor the directional movement ormigration of a suitable cell capable of chemotaxis, such as a leukocyte(e.g., T lymphocytes, NK cells, monocytes), stable transfectants ofJurkat cells, mouse L1-2 pre-B cells or of other suitable host cells,for example, into or through a barrier (e.g., endothelium, a filter)toward increased levels of an agent, from a first surface of the barriertoward an opposite second surface. Membranes or filters provideconvenient barriers, such that the directional movement or migration ofa suitable cell into or through a filter, toward increased levels of anagent, from a first surface of the filter toward an opposite secondsurface of the filter, is monitored. In some assays, the membrane iscoated with a substance to facilitate adhesion, such as ICAM-1,fibronectin or collagen.

[0127] For example, one can detect or measure the migration of cells ina suitable container (a containing means), from a first chamber into orthrough a microporous membrane into a second chamber which contains anagent to be tested, and which is divided from the first chamber by themembrane. A suitable membrane, having a suitable pore size formonitoring specific migration in response to the agent, including, forexample, nitrocellulose, polycarbonate, is selected. For example, poresizes of about 3-8 microns, and preferably about 5-8 microns can beused. Pore size can be uniform on a filter or within a range of suitablepore sizes.

[0128] To assess migration, the distance of migration into the filter,the number of cells crossing the filter that remain adherent to thesecond surface of the filter, and/or the number of cells that accumulatein the second chamber can be determined using standard techniques (e.g.,by microscopy). In one embodiment, the cells are labeled with adetectable label (e.g., radioisotope, fluorescent label, antigen orepitope label), and migration can be assessed by determining thepresence of the label adherent to the membrane and/or present in thesecond chamber using an appropriate method (e.g., by detectingradioactivity, fluorescence, immunoassay). The extent of migrationinduced by an agent can be determined relative to a suitable control(e.g., compared to background migration determined in the absence of theagent, to the extent of migration induced by a second agent (i.e., astandard), compared with migration of untransfected cells induced by theagent).

[0129] Chambers can be formed from various solids, such as plastic,glass, polypropylene, polystyrene, etc. Membranes which are detachablefrom the chambers, such as a Biocoat (Collaborative Biomedical Products)or Transwell (Costar, Cambridge, Mass.) culture insert, facilitatecounting adherent cells. In the container, the filter can be situated soas to be in contact with fluid containing cells in the first chamber,and the fluid in the second chamber. Other than the test agent oradditional ligand, inhibitor, or promoter present for the purpose of theassay, the fluid on either side of the membrane is preferably the sameor substantially similar. The fluid in the chambers can comprise proteinsolutions (e.g., bovine serum albumin, fetal calf serum, human serumalbumin) which may act to increase stability and inhibit nonspecificbinding of cells, and/or culture media.

[0130] In one embodiment, transendothelial migration is assessed. Inaddition to lower background (signal to noise ratio), transendothelialmigration models in vivo conditions in which leukocytes emigrate fromblood vessels toward chemoattractants present in the tissues at sites ofinflammation by crossing the endothelial cell layer lining the vesselwall. In this embodiment, transmigration through an endothelial celllayer assessed. To prepare the cell layer, endothelial cells can becultured on a microporous filter or membrane, optionally coated with asubstance such as collagen, fibronectin, or other extracellular matrixproteins, to facilitate the attachment of endothelial cells. Preferably,endothelial cells are cultured until a confluent monolayer is formed. Avariety of mammalian endothelial cells can are available for monolayerformation, including for example, vein, artery or microvascularendothelium, such as human umbilical vein endothelial cells (CloneticsCorp, San Diego, Calif.) or a suitable cell line, such as the ECV 304cell line used (European Collection of Animal Cell Cultures, PortonDown, Salisbury, U.K.). To assay chemotaxis in response to a particularmammalian receptor, endothelial cells of the same mammal are preferred;however endothelial cells from a heterologous mammalian species or genuscan also be used.

[0131] Generally, the assay is performed by detecting the directionalmigration of cells into or through a membrane or filter, in a directiontoward increased levels of an agent, from a first surface of the filtertoward an opposite second surface of the filter, wherein the filtercontains an endothelial cell layer on a first surface. Directionalmigration occurs from the area adjacent to the first surface, into orthrough the membrane, towards an agent situated on the opposite side ofthe filter. The concentration of agent present in the area adjacent tothe second surface, is greater than that in the area adjacent to thefirst surface.

[0132] In one embodiment, a chemotaxis assay is used to test for ligandor promoter activity of an agent, a composition comprising cells capableof migration and expressing a mammalian CXCR3 protein or functionalvariant thereof are placed in the first chamber, and a compositioncomprising the agent (one or more agents) to be tested is placed in thesecond chamber, preferably in the absence of other ligands or promoterscapable of inducing chemotaxis of the cells in the first chamber (havingchemoattractant function). However, one or more ligands or promotershaving chemoattractant function may be present. The ability of an agentto induce chemotaxis of the cells expressing a mammalian CXCR3 receptorin this assay is indicative that the agent is a ligand or promoter ofreceptor function.

[0133] In one embodiment used to test for an inhibitor, a compositioncomprising cells capable of migration and expressing a mammalian CXCR3protein or functional variant are placed in the first chamber. Acomposition comprising a ligand or promoter (i.e., one or more ligandsor promoters) capable of inducing chemotaxis of the cells in the firstchamber (having chemoattractant function) is placed in the secondchamber. Before (preferably shortly before) the cells are placed in thefirst chamber, or simultaneously with the cells, a compositioncomprising the agent to be tested is placed, preferably, in the firstchamber. The ability of an agent to inhibit ligand- or promoter-inducedchemotaxis of the cells expressing a mammalian CXCR3 protein in thisassay is indicative that the agent is an inhibitor of receptor function(e.g., an inhibitor of cellular response function). A reduction in theextent of migration induced by the ligand or promoter in the presence ofthe test agent, is indicative of inhibitory activity. Separate bindingstudies (see above) can be performed to determine whether inhibition isa result of binding of the test agent to receptor or occurs via adifferent mechanism.

[0134] In vivo assays which monitor leukocyte infiltration of a tissue,in response to injection of an agent in the tissue, are described below.These models measure the ability of cells to respond to a ligand orpromoter by emigration and chemotaxis to a site of inflammation.

[0135] The effects of a ligand, inhibitor or promoter on the cellularresponse function of a CXCR3 receptor can be assessed by monitoringother cellular responses induced by active receptor, using suitable hostcells containing receptor. Similarly, these assays can be used todetermine the function of a receptor. For instance, exocytosis (e.g.,degranulation of natural killer cells leading to release of one or moreenzymes or other granule components, such as esterases (e.g., serineesterases), perforin, and/or granzymes), inflammatory mediator release(such as release of bioactive lipids such as leukotrienes (e.g.,leukotriene C₄)), and respiratory burst, can be monitored by methodsknown in the art or other suitable methods. (See e.g., Taub, D. D. etal., J. Immunol., 155: 3877-3888 (1995), regarding assays for release ofgranule-derided serine esterases (the teachings of which areincorporated herein by reference) and Loetscher et al., J. Immunol.,156: 322-327 (1996), regarding assays for enzyme and granzyme release byNK cells and cytotoxic T lymphocytes (CTLs) (the teachings of which areincorporated herein by reference); Rot, A. et al., J. Exp. Med., 176:1489-1495 (1992) regarding respiratory burst; Bischoff, S. C. et al.,Eur. J. Immunol., 23: 761-767 (1993) and Baggliolini, M. and C. A.Dahinden, Immunology Today, 15: 127-133 (1994)).

[0136] In one embodiment, a ligand, inhibitor and/or promoter isidentified by monitoring the release of an enzyme upon degranulation orexocytosis by a cell capable of this function. Cells containing anucleic acid of the present invention, which encodes an active receptorprotein capable of stimulating exocytosis or degranulation aremaintained in a suitable medium under suitable conditions, wherebyreceptor is expressed and degranulation can be induced. The receptor iscontacted with an agent to be tested, and enzyme release is assessed.The release of an enzyme into the medium can be detected or measuredusing a suitable assay, such as in an immunological assay, orbiochemical assay for enzyme activity.

[0137] The medium can be assayed directly, by introducing components ofthe assay (e.g., substrate, co-factors, antibody) into the medium (e.g.,before, simultaneous with or after the cells and agent are combined).Alternatively, the assay can be performed on medium which has beenseparated from the cells or further processed (e.g., fractionated) priorto assay. For example, convenient assays for are available for enzymessuch serine esterases (see e.g., Taub, D. D. et al., J. Immunol., 155:3877-3888 (1995) regarding release of granule-derived serine esterases).

[0138] Stimulation of degranulation by an agent can be indicative thatthe agent is a ligand or promoter of a mammalian CXCR3 protein. Inanother embodiment, cells expressing receptor are combined with a ligandor promoter, and an agent to be tested is added before, after orsimultaneous therewith, and degranulation is assessed. Inhibition ofligand- or promoter-induced degranulation is indicative that the agentis an inhibitor of mammalian CXCR3 protein function.

[0139] Cellular adherence can also monitored by methods known in the artor other suitable methods. Engagement of the chemokine receptors of alymphocyte can cause integrin activation, and induction of adherence toadhesion molecules expressed in vasculature or the perivascular space.In one embodiment, a ligand, inhibitor and/or promoter is identified bymonitoring cellular adherence by a cell capable of adhesion. Forexample, an agent to be tested can be combined with (a) cells expressingreceptor (preferably non-adherent cells which when transfected withreceptor aquire adhesive ability), (b) a composition comprising asuitable adhesion molecule (e.g., a substrate such as a culture wellcoated with an adhesion molecule, such as fibronectin), and (c) a ligandor promoter (e.g., agonist), and maintained under conditions suitablefor ligand- or promoter-induced adhesion. Labeling of cells with afluorescent dye provides a convenient means of detecting adherent cells.Nonadherent cells can be removed (e.g., by washing) and the number ofadherent cells determined. The effect of the agent in inhibiting orenhancing ligand- or promoter-induced adhesion can be indicative ofinhibitor or promoter activity, respectively. Agents active in the assayinclude inhibitors and promoters of binding, signalling, and/or cellularresponses. In another embodiment, an agent to be tested can be combinedwith cells expressing receptor and a composition comprising a suitableadhesion molecule under conditions suitable for ligand- orpromoter-induced adhesion, and adhesion is monitored. Increased adhesionrelative to a suitable control is indicative of the presence of a ligandand/or promoter.

[0140] Models of Inflammation

[0141] A variety of in vivo models of inflammation are available, whichcan be used to assess the effects of ligands, inhibitors, or promotersin vivo as therapeutic agents, including a sheep model for asthma (seee.g., Weg, V. B. et al., J. Exp. Med., 177: 561 (1993), the teachings ofwhich are incorporated herein by reference), a rat delayed typehypersensitivity model (Rand, M. L. et al., Am. J. Pathol., 148: 855-864(1996), the teachings of which are incorporated herein by reference), orother suitable models. The activity of antibodies which cross-react withother mammalian CXCR3 proteins can be assessed in such mammals.

[0142] In addition, leukocyte infiltration upon intradermal injection ofa compound into a suitable animal, such as rabbit, rat, or guinea pig,can be monitored (see e.g., Van Damme J. et al., J. Exp. Med., 176:59-65 (1992); Zachariae, C. O. C. et al., J. Exp. Med., 171: 2177-2182(1990); Jose, P. J. et al., J. Exp. Med., 179: 881-887 (1994)). In oneembodiment, skin biopsies are assessed histologically for infiltrationof leukocytes (e.g., T lymphocytes, monocytes, natural killer cells). Inanother embodiment, labeled cells (e.g., cells expressing a mammalianCXCR3 protein which are labeled with ¹¹¹In, for example) capable ofchemotaxis and extravasation are administered to the animal.Infiltration of labelled cells in the vicinity of the site of injectionof a test sample (e.g., a compound to be tested in a suitable buffer orphysiological carrier) is indicative of the presence of a ligand orpromoter, such as an agonist, in the sample. These assays can also bemodified to identify inhibitors of chemotaxis and leukocyteextravasation. For example, an inhibitor can be administered, eitherbefore, simultaneously with or after ligand or agonist is administeredto the test animal. A decrease of the extent of infiltration in thepresence of inhibitor as compared with the extent of infiltration in theabsence of inhibitor is indicative of inhibition.

[0143] Diagnostic Applications

[0144] The present invention has a variety of diagnostic applications.For example, a mutation(s) in a gene encoding a mammalian CXCR3 proteincan cause a defect in at least one function of the encoded receptor,thereby reducing or enhancing receptor function. For instance, amutation which produces a variant of receptor or alters the level ofexpression, can reduce or enhance receptor function, reducing orenhancing processes mediated by receptor (e.g., inflammatory processes).The presence of such a mutation can be determined using methods whichdetect or measure the presence of receptor or receptor function in cells(e.g., leukocytes, such as activated T lymphocytes) of an individual orin a receptor preparation isolated from such cells. In these assays,reduced or enhanced levels of receptor and/or reduced or enhancedreceptor function can be assessed.

[0145] The nucleic acids of the present invention provide reagents, suchas probes and PCR primers, which can be used to screen for, characterizeand/or isolate a defective mammalian CXCR3 gene, which encodes areceptor having reduced or enhanced activity. Standard methods ofscreening for a defective gene can be employed, for instance. Adefective gene can be isolated and expressed in a suitable host cell forfurther assessment as described herein for mammalian CXCR3 proteins. Anumber of human diseases are associated with defects in the function ofa G-protein coupled receptor (Clapham, D. E., Cell, 75: 1237-1239(1993); Lefkowitz, R. J., Nature, 365: 603-04 (1993)).

[0146] The nucleic acids of the present invention provide reagents, suchas probes and PCR primers, which can also be used to assess expressionof receptor (e.g., by detecting transcription of mRNA) by cells in asample (e.g., by Northern analysis, by in situ hybridization). Forexample, expression in activated T lymphocytes or other cell types canbe assessed.

[0147] The antibodies of the present invention have application inprocedures in which receptor can be detected on the surface of cells.The receptor provides a marker of the leukocyte cell types in which itis expressed, particularly of activated T cells. For example, antibodiesraised against a receptor protein or peptide, such as the antibodiesdescribed herein (e.g., mAb 1C6), can be used to detect and/or quantifycells expressing receptor. In one embodiment, the antibodies can be usedto sort cells which express receptor from among a mixture of cells(e.g., to isolate activated T cells, such as CD4⁺ T cells). Suitablemethods for counting and/or sorting cells can be used for this purpose(e.g., flow cytometry, fluorescence activated cell sorting). Cell countscan be used in the diagnosis of diseases or conditions in which anincrease or decrease in leukocyte cell types (e.g., activated T cells)is observed. The presence of an increased level of activated T cells ina sample obtained from an individual can be indicative of infiltrationdue to an inflammatory disease or condition, such as a delayed typehypersensitivity reaction, allograft rejection, or a pathologiccondition, including bacterial or viral infection.

[0148] Furthermore, the antibodies can be used to detect or measureexpression of receptor. For example, antibodies of the present inventioncan be used to detect or measure receptor in a sample (e.g., tissues orbody fluids from an individual such as blood, serum, leukocytes (e.g.,activated T lymphocytes), bronchoalveolar lavage fluid, saliva, bowelfluid). For example, a sample (e.g., tissue and/or fluid) can beobtained from an individual and a suitable assay can be used to assessthe presence or amount of CXCR3 protein. Suitable assays includeimmunological methods such as FACS analysis and enzyme-linkedimmunosorbent assays (ELISA), including chemiluminescence assays,radioimmunoassay, and immunohistology. Generally, a sample and antibodyof the present invention are combined under conditions suitable for theformation of an antibody-receptor complex, and the formation ofantibody-receptor complex is assessed (directly or indirectly).

[0149] The presence of an increased level of receptor reactivity in asample obtained from an individual can be indicative of inflammationand/or leukocyte (e.g., activated T cell) infiltration and/oraccumulation associated with an inflammatory disease or condition, suchas allograft rejection, delayed type hypersensitivity reaction, or aninfection such as a viral or bacterial infection. The level ofexpression of a mammalian CXCR3 protein or variant can also be used tocorrelate increased or decreased expression of a mammalian CXCR3 proteinwith a particular disease or condition, and in the diagnosis of adisease or condition in which increased or decreased expression of amammalian CXCR3 protein occurs (e.g., increased or decreased relative toa suitable control, such as the level of expression in a normalindividual). Similarly, the course of therapy can be monitored byassessing CXCR3 immunoreactivity in a sample from a patient. Forexample, antibodies of the present invention can be used to monitor thenumber of cells bearing CXCR3 in a sample (e.g., blood, tissue) from apatient being treated with an anti-inflammatory or immunosuppressiveagent

[0150] Transgenic Animals

[0151] Transgenic animals, in which the genome of the animal host isaltered using recombinant DNA techniques, can be constructed. In oneembodiment, the alteration is not heritable (e.g., somatic cells, suchas progenitor cells in bone marrow, are altered). In another embodiment,the alteration is heritable (the germ line is altered). Transgenicanimals can be constructed using standard techniques or other suitablemethods (see e.g., Cooke. M. P. et al., Cell, 65: 281-291 (1991)regarding alteration of T lymphocytes; Hanahan, D., Science, 246:1265-1275, (1989); Anderson et al., U.S. Pat. No. 5,399,346).

[0152] In one aspect, an endogenous mammalian CXCR3 gene can beinactivated or disabled, in whole or in part, in a suitable animal host(e.g., by gene disruption techniques) to produce a transgenic animal.Nucleic acids of the present invention can be used to assess successfulconstruction of a host containing an inactivated or disabled CXCR3 gene(e.g., by Southern hybridization). In addition, successful constructionof a host containing an inactivated or disabled CXCR3 gene can beassessed by suitable assays which monitor the function of the encodedreceptor. Such animals can be used to assess the effect of receptorinactivation on inflammation and host defenses against cancer andpathogens (e.g., a viral pathogen).

[0153] In another embodiment, a nucleic acid encoding a mammalian CXCR3protein or polypeptide is introduced into a suitable host to produce atransgenic animal. In a preferred embodiment, endogenous CXCR3 receptorgenes present in the transgenic animals are inactivated (e.g.,simultaneously with introduction of the nucleic acid by homologousrecombination, which disrupts and replaces the endogenous gene). Forexample, a transgenic animal (e.g., a mouse, guinea pig, sheep) capableof expressing a nucleic acid encoding a mammalian CXCR3 receptor of adifferent mammalian species (e.g., a human CXCR3 such as the CXCR3encoded by SEQ ID NO:1) in leukocytes (such as lymphocytes (e.g.,activated T lymphocytes), natural killer cells) can be produced, andprovides a convenient animal model far assessing the function of theintroduced receptor. In addition, a test agent can be administered tothe transgenic animal, and the effect of the agent on areceptor-mediated process (e.g., inflammation) can be monitored asdescribed herein or using other suitable assays. In this manner, agentswhich inhibit or promote receptor function can be identified or assessedfor in vivo effect.

[0154] Methods of Therapy

[0155] Modulation of mammalian CXCR3 function according to the presentinvention, through the inhibition or promotion of at least one functioncharacteristic of a mammalian CXCR3 protein, provides an effective andselective way of inhibiting or promoting receptor-mediated functions. AsCXC chemokine receptors selectively expressed on activated lymphocytes,responsive to chemokines such as IP-10 and Mig whose primary targets arelymphocytes, particularly effector cells such as activated or stimulatedT lymphocytes and NK cells, mammalian CXCR3 proteins provide a targetfor selectively interfering with or promoting lymphocyte function in amammal, such as a human. Once lymphocytes are recruited to a site, otherleukocyte types, such as monocytes, may be recruited by secondarysignals. Thus, agents which inhibit or promote CXCR3 function, includingligands, inhibitors (e.g., 1C6) and/or promoters, such as thoseidentified as described herein, can be used to modulate leukocytefunction (e.g., leukocyte infiltration including recruitment and/oraccumulation) particularly of lymphocytes, for therapeutic purposes.

[0156] In one aspect, the present invention provides a method ofinhibiting or promoting an inflammatory response in an individual inneed of such therapy, comprising administering an agent which inhibitsor promotes mammalian CXCR3 function to an individual in need of suchtherapy. In one embodiment, a compound which inhibits one or morefunctions of a mammalian CXCR3 protein (e.g., a human CXCR3) isadministered to inhibit (i.e., reduce or prevent) inflammation. Forexample, antibodies of the present invention, including mAb 1C6 can beused in the method. As a result, one or more inflammatory processes,such as leukocyte emigration, chemotaxis, exocytosis (e.g., of enzymes)or inflammatory mediator release, is inhibited. For example, leukocyticinfiltration of inflammatory sites (e.g., in a delayed-typehypersensitivity response) can be inhibited according to the presentmethod.

[0157] In another embodiment, an agent (e.g., receptor agonist) whichpromotes one or more functions of a mammalian CXCR3 protein (e.g., ahuman CXCR3) is administered to induce (trigger or enhance) aninflammatory response, such as leukocyte emigration, chemotaxis,exocytosis (e.g., of enzymes) or inflammatory mediator release,resulting in the beneficial stimulation of inflammatory processes. Forexample, natural killer cells can be recruited to combat viralinfections or neoplastic disease.

[0158] The term “individual” is defined herein to include animals suchas mammals, including, but not limited to, primates (e.g., humans),cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, miceor other bovine, ovine, equine, canine, feline, rodent or murinespecies. Diseases and conditions associated with inflammation,infection, and cancer can be treated using the method. In a preferredembodiment, the disease or condition is one in which the actions oflymphocytes, particularly effector cells such as activated or stimulatedT lymphocytes and natural killer (NK) cells, are to be inhibited orpromoted for therapeutic (including prophylactic) purposes. In aparticularly preferred embodiment, the inflammatory disease or conditionis a T cell-mediated disease or condition.

[0159] Diseases or conditions, including chronic diseases, of humans orother species which can be treated with inhibitors of CXCR3 function,include, but are not limited to:

[0160] inflammatory or allergic diseases and conditions, includingsystemic anaphylaxis or hypersensitivity responses, drug allergies(e.g., to penicillin, cephalosporins), insect sting allergies;inflammatory bowel diseases, such as Crohn's disease, ulcerativecolitis, ileitis and enteritis; vaginitis; psoriasis and inflammatorydermatoses such as dermatitis, eczema, atopic dermatitis, allergiccontact dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous,and hypersensitivity vasculitis); spondyloarthropathies; scleroderma;respiratory allergic diseases such as asthma, allergic rhinitis,hypersensitivity lung diseases, hypersensitivity pneumonitis,interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis,or ILD associated with rheumatoid arthritis, or other autoimmuneconditions);

[0161] autoimmune diseases, such as arthritis (e.g., rheumatoidarthritis, psoriatic arthritis), multiple sclerosis, systemic lupuserythematosus, myasthenia gravis, diabetes, including diabetes mellitusand juvenile onset diabetes, glomerulonephritis and other nephritides,autoimmune thyroiditis, Behcet's disease;

[0162] graft rejection (e.g., in transplantation), including allograftrejection or graft-versus-host disease;

[0163] other diseases or conditions in which undesirable inflammatoryresponses are to be inhibited can be treated, including, but not limitedto, atherosclerosis, cytokine-induced toxicity, myositis (includingpolymyositis, dermatomyositis).

[0164] Diseases or conditions of humans or other species which can betreated with promoters (e.g., an agonist) of CXCR3 function, include,but are not limited to:

[0165] cancers, particularly those with leukocytic infiltration of theskin or organs such as cutaneous T cell lymphoma (e.g., mycosisfungoides);

[0166] diseases in which angiogenesis or neovascularization plays arole, including neoplastic disease, retinopathy (e.g., diabeticretinopathy), and macular degeneration;

[0167] infectious diseases, such as bacterial infections and tuberculoidleprosy, and especially viral infections;

[0168] immunosuppression, such as that in individuals withimmunodeficiency syndromes such as AIDS, individuals undergoingradiation therapy, chemotherapy, or other therapy which causesimmunosuppression; immunosuppression due congenital deficiency inreceptor function or other causes. Promoters of CXCR3 function can alsohave protective effects useful to combat stem cell depletion duringcancer chemotherapy (Sarris, A. H. et al., J. Exp. Med., 178: 1127-1132(1993)).

[0169] Modes of Administration

[0170] According to the method, one or more agents can be administeredto the host by an appropriate route, either alone or in combination withanother drug. An effective amount of an agent (e.g., a receptor peptidewhich inhibits ligand binding, an anti-CXCR3 antibody or antigen-bindingfragment thereof) is administered. An effective amount is an amountsufficient to achieve the desired therapeutic or prophylactic effect,under the conditions of administration, such as an amount sufficient forinhibition or promotion of CXCR3 receptor function, and thereby,inhibition or promotion, respectively, of a receptor-mediated process(e.g., an inflammatory response).

[0171] A variety of routes of administration are possible including, butnot necessarily limited to oral, dietary, topical, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection), andinhalation (e.g., intrabronchial, intranasal or oral inhalation,intranasal drops) routes of administration, depending on the agent anddisease or condition to be treated. For respiratory allergic diseasessuch as asthma, inhalation is a preferred mode of administration.

[0172] Formulation of an agent to be administered will vary according tothe route of administration selected (e.g., solution, emulsion,capsule). An appropriate composition comprising the agent to beadministered can be prepared in a physiologically acceptable vehicle orcarrier. For solutions or emulsions, suitable carriers include, forexample, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils, for instance.Intravenous vehicles can include various additives, preservatives, orfluid, nutrient or electrolyte replenishers and the like (See,generally, Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Co., PA, 1985). For inhalation, the agent can be solubilizedand loaded into a suitable dispenser for administration (e.g., anatomizer, nebulizer or pressurized aerosol dispenser).

[0173] Furthermore, where the agent is a protein or peptide, the agentcan be administered via in vivo expression of the recombinant protein.In vivo expression can be accomplished via somatic cell expressionaccording to suitable methods (see, e.g. U.S. Pat. No. 5,399,346). Inthis embodiment, nucleic acid encoding the protein can be incorporatedinto a retroviral, adenoviral or other suitable vector (preferably, areplication deficient infectious vector) for delivery, or can beintroduced into a transfected or transformed host cell capable ofexpressing the protein for delivery. In the latter embodiment, the cellscan be implanted (alone or in a barrier device), injected or otherwiseintroduced in an amount effective to express the protein in atherapeutically effective amount.

[0174] Exemplification

[0175] The present invention will now be illustrated by the followingExamples, which are not intended to be limiting in any way.

[0176] Human Chemokines (Examples 1-2)

[0177] The CXC chemokines Mig, IL-8, GROα, NAP-2, GCP-2, ENA78, PF4, theCC chemokines MCP-1, MCP-2, MCP-3, MCP-4, MIP-1α, MIP-1β, RANTES, I309,eotaxin and the chemokine-related lymphotactin were chemicallysynthesized according to established protocols (Clark-Lewis, I. et al.,Biochemistry 30: 3128-3135 (1991)). The CXC chemokine IP-10 waspurchased from PeproTech, Rocky Hill, N.J.

EXAMPLE 1 Cloning of Receptor cDNA

[0178] Standard molecular biology techniques were used (Sambrook, J. etal., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY).

[0179] DNA fragments coding for putative T lymphocyte-restrictedchemokine receptors were generated using the polymerase chain reaction(PCR). Two degenerate oligonucleotide primers were designed based onconserved motifs of chemokine receptors. Primer design was based on theconserved nucleotide sequences within transmembrane domain 2 (TM2) andtransmembrane domain 7 (TM7) of the chemokine receptors IL-8RI (CXCR1),IL-8R2 (CXCR2), CC-CKR1 (CCR1), CC-CKR2 (CCR2) and the orphan receptorsEBI I, LESTR, and BLR1/MDR15 (EBI I, Birkenbach, M. et al., J. Virol.,67: 2209-2220 (1993)); LESTR, Loetscher, M. et al., J. Biol. Chem., 269:232-237 (1994); and BLR1/MDR15, Dobner, T. et al., Eur. J. Immunol., 22:2795-2799 (1992) and Barella, L. et al., Biochem. J., 309: 773-779(1995)).

[0180] The sequences of the primers were as follows;

[0181] SEQ ID NO:3:

[0182] 5′-GGG CTG CAG CII T(T/G) (T/G) C(C/A)G AC(A/C) TIC TI(C/T) T-3′

[0183] SEQ ID NO:4:

[0184] 5′-GGG TCT AGA IGG GTT IAI (G/A)CA (G/A)C(T/A) (G/A) (T/C)G-3′

[0185] (I=inosine). These primers were used in a polymerase chainreaction (PCR) to amplify DNA fragments using human genomic DNA isolatedfrom human peripheral blood lymphocytes as template as follows. A 100 μlreaction mixture containing 2 μg human genomic DNA, 1×DynaZyme buffer(Finnzymes OY, Espoo, Finland), 1.5 mM MgCl₂₁ 500 μM of eachdeoxynucleotide, 1 μM of both primers, and 25 U of DynaZyme DNApolymerase was subjected to 30 cycles (94° C. for 1 minute; 55° C. for 1minute; and 72° C. for 2 minutes) on a DNA thermal cycler (Techne PHC-2,Brouwer, Switzerland) PCR products of the predicted size (approximately700 bp) were cloned into the Gene Scribe-Z vectors pTZ18/19 UT/R (USB,Cleveland, OH), were partially sequenced (Sanger, F. et al., Proc. Natl.Acad. Sci. USA, 74: 5463-5467 (1977)), and were evaluated for theirsimilarity to known chemokine receptors and for expression of theircorresponding mRNA in leukocytes. A DNA fragment designated 2MLC22revealed 640 nucleotide sequence identity with IL-8R2. Fragment 2MLC22specifically hybridized to RNA from T cells, but not monocytes orneutrophils, as assessed by Northern blot analysis using a hybridizationprobe prepared by enzymatically labeling 2MLC22 with the radioactiveisotope ³²P using Klenow fragment of DNA Polymerase I and a commerciallyavailable random-prime labeling kit.

[0186] Fragment 2MLC22 was enzymatically labeled with 32p as describedand used as a probe to screen a human tetanus toxoid-specific CD4⁺ Tcell (KT30) cDNA library, prepared in lambda-ZAP Express (Stratagene,Zurich, Switzerland) (Loetscher, M. et al., J. Bio. Chem., 269: 232-237(1994)). A cDNA library was prepared in a λ ZAP Express system accordingto the manufacturer's protocol (Stratagene GMBH, Zurich, Switzerland)using poly(A)⁺ RNA from human tetanus toxoid-specific CD4⁺ T cells(KT30). The resulting library contained about 1.8×10⁶ independent cloneswith an average insert size of approximately 1.1 kb. For plaquehybridization screening, about 4×10⁵ clones were transferred ontoBiodyne nylon membranes (PALL AG, Mittenz, Switzerland) and probed with2MLC22 which had been labeled to a specific activity of 1×10⁹ dpm/μg DNAusing the high prime DNA labeling kit (Boehringer Mannheim, Mannheim,Germany). Hybridization was carried out in 50% formamide, 6×SSC, 0.5%SDS, 100 μg/ml denatured salmon sperm DNA at 42° C. for 20 hours using1×10⁶ dpm 2MLC22/ml hybridization solution. The membranes were washedonce in 2×SSC, 0.1% SDS at room temperature for 10 minutes, twice in1×SSC, 0.1% SDS at 65° C. for 30 minutes, and finally once in 0.5×SSC,0.1% SDS at 650° C. for 10 minutes. Twenty-three clones were isolatedfrom hybridization positive lambda plaques following the high stringencywashes, and the clone with the largest insert (1670 bp) was sequenced.CXCR3 cDNA was subcloned into commercially available plasmid vectors fornucleotide sequencing, generation of hybridization probes, andconstruction of stably transfected mammalian cell clones expressingCXCR3, and these CXCR3 cDNA-containing constructs are maintained in E.coli strains.

[0187] Results

[0188] A cDNA was isolated from a human CD4⁺ T cell library by searchingfor T lymphocyte-specific chemokine receptors (FIG. 1, SEQ ID NO:1).This cDNA was not recovered in the course of searching a commonly usedmonocyte-derived cDNA library or granulocyte (HL60)-derived cDNA libraryfor novel chemokine receptor cDNAs; however, a direct search of thelibraries specifically for CXCR3 cDNA has not been conducted. The CXCR3cDNA, which was shown to encode an IP-10/Mig receptor (see below), andhas an open reading frame (ORF) of 1104 bp beginning at residue 69 whichencodes a protein of 368 amino acids with a predicted molecular mass of40,659 daltons. The amino acid sequence (FIG. 2, SEQ ID NO:2) includesseven putative transmembrane segments, which are characteristic ofG-protein coupled receptors, and three potential N-glycosylation sites(Asn²², Asn³², and Asn¹⁹⁹) (FIG. 2). In addition, one threonine and nineserine residues, which are potential phosphorylation sites for receptorkinases (Palczewski, K. and J. L. Benovic, Trends Biochem. Sci., 16:387-391 (1991); Chuang, T. T. et al., J. Biol. Chem., 267: 6886-6892(1992); and Giannini, E. et al., J. Biol. Chem., 270: 19166-19172(1995)), can be found in the intracellular COOH-terminal region (FIG.2).

[0189] The 368 amino acid sequence of the receptor (IP-10/MigR, FIG. 2,SEQ ID NO:2) was aligned with the amino acid sequences of other humanchemokine receptors, including IL-8R1 (CXCR1), IL-8R2 (CXCR2), CC-CKR1(CCR1), CC-CKR2A (CCR2a), CC-CKR3 (CCR3) and CC-CKR4 (CCR4). Multipleprotein alignment was performed according to Higgins and Sharp (Higgins,D. G. and P. M. Sharp, “Description of the method used in CLUSTAL,”Gene, 73: 237-244 (1988)). Residues framed in black in FIG. 2 representregions of identity between IP-10/MigR and at least two other chemokinereceptors. Hyphens indicate gaps in the alignment. The alignmentrevealed several conserved motifs, particularly in the transmembranedomains and the second intracellular loop. Significant sequence identitywith CXC receptors IL-8R1 and IL-8R2, but not with the CC chemokinereceptors, was observed in the third and the sixth transmembrane domains(FIG. 2).

[0190] The sequence shares 40.9% and 40.3i amino acid identity overallwith the IL-8R1 and IL-8R2 receptors, respectively, and 34.2 to 36.9%identity with the five known CC chemokine receptors (Table 1). A lowerdegree of similarity was found with seven-transmembrane-domain receptorsthat are expressed in T cells, but which do not bind chemokines, e.g.,27.2% identity with the thrombin receptor (Vu, T. -K. H. et al., Cell,64: 1057-1068 (1199)). A truncated clone of unidentified function, withan incomplete coding sequence which can be aligned with that of FIG. 2,was previously isolated from a human genomic DNA library (Marchese, A.et al., Genomics, 29: 335-344 (1995)). TABLE 1 Amino Acid SequenceComparison of IP-10/MigR with Human Chemokine Receptors IL-8R1 IL-8R2CC-CKR1 CC-CKR2A CC-CKR3 CC-CKR4 CC-CKR5 (CXCR1) (CXCR2) (CCR1) (CCR2a)(CCR3) (CCR4) (CCR5) ThrombR IP-10/MigR 40.9^(a) 40.3 34.9 34.2 34.435.8 36.9 27.2 IL-8R1 77.1 33.7 32.9 34.3 39.7 34.3 29.1 IL-8R2 34.933.6 34.1 40.8 34.4 29.7 CC-CKR1 54.1 63.1 49.3 56.3 26.8 CC-CKR2A 50.746.1 68.8 24.6 CC-CKR3 46.5 52.3 27.3 CC-CKR4 50.0 29.2 CC-CKR5 23.6

EXAMPLE 2 Biological Activity

[0191] Expression in Activated T Lymphocytes

[0192] In view of the observed chemokine selectivity, the occurrence ofthe IP-10/MigR in leukocytes and related cell lines was examined byNorthern blot analysis. 10 μg samples of total RNA were examined fromfreshly isolated human blood monocytes, neutrophils, lymphocytes (PBL),nylon-wool purified T cells, and from cultured cells including clonedhuman CD4⁺ T cells (KT30) and CD8⁺ T cells (ERCD8), cloned NK cells(ERNK57), and PBL cultured for 10 days (1-2.5×10⁶ cells/ml in RPMI 1640medium containing 2 mM glutamine, 1×non-essential amino acids, 1 mMsodium pyruvate, 100 μg/ml kanamycin, 5×10⁻⁵ M 2-mercaptoethanol, and 5%human serum) in the presence of 400 U/ml hrIL-2. (human recombinant IL-2was a gift of Dr. A. Lanzavecchia, Basel Institute of Immunology, Basel,Switzerland). Agarose gels were stained with ethidium bromide to checkthe integrity and amount of total RNA on the gel prior to blotting. RNAsamples were analyzed with ³²P-labeled 5′-fragment of the IP-10/MigR DNA(10⁹ cpm/μg DNA) at 5×10⁶ cpm/ml hybridization solution as described(Loetscher, M. et al., J. Biol. Chem., 269: 232-237 (1994)). The5′-fragment used as a Northern probe was prepared by digestion of CXCR3cDNA in pBK-CMV vector (Stratagene GMBH, Zurich, Switzerland) with PstIyielding the 724 bp 5′-end of the CXCR3 cDNA (FIG. 1).

[0193] Results

[0194] Abundant expression of mRNA of the expected size was found in thecloned CD4⁺ T cells, KT30, that were used for isolation of the receptorcDNA. Similar levels of expression were observed in the CD8⁺ T cellclone, EP-CD8, and the NK cell clone, ERNK57. In contrast, in freshlyisolated blood lymphocytes and nylon-wool purified T cells, IL10/MigRtranscripts were barely detectable. However, when these cells werecultured in the presence of IL-2, a strong upregulation was obtained,and the level of receptor mRNA approached that of T and NK cell clones.No IP-10/MigR transcripts were detected under these conditions infreshly isolated blood monocytes, neutrophil leukocytes, or eosinophilleukocytes. Additional leukocyte-related cells that did not expressIP-10/MigR mRNA include the mast cell line, HMC-1, the promyelocyticleukemia line, HL60, the histiocytic lymphoma, U937, the chronicmyelogenous leukemia line, K562, the acute T cell leukemia line, Jurkat,the acute lymphoblastic leukemia line, Molt, the B-lymphoblastic celllines Daudi and Raji, lymphocytes from patients with chronic and acuteB-lymphoid leukemia (B-CLL and B-ALL), mature basophils from a patientwith basophilic leukemia, and the erythroleukemia cell line, HEL. Bycontrast, the receptors for chemokines which have been shown previouslyto attract lymphocytes, i.e. MCP-1 MCP-2, MCP-3, MIP-1, MIP-10 andRANTES (Loetscher, P. et al., FASEB J., 8: 1055-1060 (1994); Carr, M. W.et al., Proc. Natl. Acad. Sci. USA 91: 3652-3656 (1994); Taub, D. D. etal., Science, 260: 355-358 (1993); Schall, T. J. et al., J. Exp. Med.,177: 1821-1825 (1993); Schall, T. J. et al., Nature, 347: 669-672(1990)), are also found in monocytes and granulocytes. The restrictedexpression of ID-10/MigR in activated T lymphocytes and a natural killercell line suggests that this novel receptor can mediate selectivelymphocyte recruitment.

[0195] Stable Transfectants

[0196] CXCR3 cDNA was released from pBK-CMV (Stratagene GMBH, Zurich,Switzerland) by digestion with BamHI and XbaI, and was cloned into BamHIand XbaI sites of pcDNA3 (Invitrogen BV, WB Leek, Netherlands) to yieldpcDNA3-Clone8, which is maintained and stored Escherichia coli(XL1Blue).

[0197] To generate stable transfectants, 4×10⁶ of either mouse pre-Bcells (300-19) (Thelen, M. et al., FASEB. J., 2: 2702-2706 (1988)),human promyelocytic cells (GM-1) (Garotta, G. et al., J. LeukocyteBiol., 49: 294-301 (1991)) or human acute T cell leukemia cells (Jurkat)(Loetscher, P. et al., FEBS Lett. 341: 187-192 (1994)), were transfectedby electroporation with 20 μg of receptor cDNA in pcDNA3 which waslinearized with Bgl II as described previously (Moser, B. et al.,Biochem. J., 294: 285-292 (1993)).

[0198] IP-10/MigR transfected cells were cloned by limiting dilutionunder G-418 (Life Technologies, Inc.) selection (1.0 mg/ml G-418 for300-19 and 0.8 mg/ml G-418 for Jurkat and GM-1 cells). G-418 resistantclones were screened for receptor expression by RNA Dot-blot analysis.

[0199] Ca²⁺ Flux

[0200] To determine whether the receptor was functional, clones ofmurine pre-B cells (300-19), human promyelccytic cells (GM-1), and humanT cell leukemia cells (Jurkat) were stably transfected with receptorcDNA as described above. Activation of chemokine receptors leads to atransient rise in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_(i)),and this assay was used to monitor signalling in the transfected cells.

[0201] Changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_(i))were measured in cells loaded with fura-2 by incubation for 30 minutesat 37° C. with 0.1 nmol fura-2 acetoxymethylester per 106 cells in abuffer containing 136 mM NaCl, 4.8 mM KCl, 1 mM CaCl₂, 5 mM glucose, and20 mM HEPES, pH 7.4. After centrifugation, loaded cells were resuspendedin the same buffer (10⁶ cells/ml), stimulated with the indicatedchemokine at 37° C., and the [Ca²⁺]_(i)-related fluorescence changeswere recorded (von Tscharner, V. et al., Nature, 324: 69-372 (1986)).

[0202] Results

[0203] A rapid [Ca²⁺]_(i) rise was observed in response to IP-10 andMig. The chemokine IP-10 has been shown to be expressed in cutaneousdelayed-type hypersensitivity reactions (Luster, A. D. et al., Nature,315: 672-676 (1985); Kaplan, G. et al., J. Exp. Med., 166: 1098-1108(1987)). The chemokine designated Mig was recently identified (Farber,J. M., Proc. Natl. Acad. Sci. USA, 87: 5238-5242 (1990); Farber, J. M.,Biophys. Res. Commun., 192: 223-230 (1993)). Both chemokines have theCXC arrangement of the first two cysteines like IL-8, but are notchemotactic for neutrophil leukocytes. It was recently reported thatIP-10 attracts T lymphocytes (Luster, A. D. and P. Leder, J. Exp. Med.,178: 1057-1065 (1993); Taub, D. D. et al., J. Exp. Med., 177: 1809-1814(1993)), and that Mig is chemotactic for tumor-associated lymphocytes(Liao, F. et al., J. Exp. Med., 182: 1301-1314 (1995)).

[0204] FIGS. 3A-3C summarize the effects of IP-10 and Mig on cellstransfected with the cDNA and expressing the functional IP-10/MigR. Asshown by the [Ca²⁺]_(i) changes (FIG. 3A), the action of IP-10 and Migwas concentration dependent and already detectable at 1 nM, indicatingthat both chemokines have high affinity for the novel receptor. TheIP-10/MigR transfectants, by contrast, did not respond to any of 16other potential agonists at concentrations up to 100 nM, including theCXC chemokines IL-8, GROα, NAP-2, GCP-2, ENA78, PF4, the CC chemokinesMCP-1, MCP-2, MCP-3, MCP-4, MIP-1α, MIP-1β, RANTES, I309, eotaxin or thechemokine related lymphotactin (not shown). Identical results wereobtained with the murine and the human transfected cells. Theseobservations demonstrate that the novel receptor is highly selective forIP-10 and Mig. Accordingly, the receptor is referred to herein as anIP-10/Mig receptor (IP-10/MigR), or as “CXCR3”, reflecting itsspecificity for CXC chemokines.

[0205] As shown in FIG. 3B, repeated stimulation with IP-10 or Migresulted in desensitization typical of chemokine receptors. Furthermore,cross-desensitization occurred when the cells were stimulated with IP-10followed by Mig or vice versa, confirming that the receptor has highaffinity for both chemokines. At 100 nM concentration, it became evidentthat Mig was more potent in cross-desensitization than IP-10, suggestinghigher affinity or binding stability of the IP-10/Mig receptor for Mig.

[0206] While expression of functional IP-10/MigR was demonstrated,binding experiments using radioactive ligands revealed non-specificbinding between 60 and 80% of the total, preventing determination ofbinding parameters. Since IP-10 and Mig are highly cationic (pI valuesof 10.8 and 11.1), nonspecific interaction with cell surfaceproteoglycans may explain these results. Indeed, chemokinereceptor-unrelated, heparinase-sensitive binding sites for IP-10 (andPF4) have been detected on a variety of blood and tissue cells (Luster,A. D. et al., J. Exp. Med., 182: 219-231 (1995)), and heparan sulfatebinds IP-10 and Mig and prevents lymphocyte chemotaxis (not shown). Theheparin binding site is probably not involved in CXCR3 receptor binding,and inclusion of a suitable heparin derivative such as chondroitinsulfate in the reaction (e.g., in binding buffer) can be used to inhibitnon-specific binding to cells through the heparin binding site.

[0207] Chemotaxis

[0208] PBL were freshly isolated from donor blood buffy coats. Donorblood buffy coats were provided by the Swiss Central Laboratory BloodTransfusion Service, SRK. Isolation of buffy coat PBL was performed asdescribed in Colotta, F. et al., J. Immunol., 132: 936-944 (1984)Freshly isolated PBL from donor blood buffy coats were used withoutfurther processing, or were used after culturing for 10 days in thepresence of IL-2 (1-2.5×10⁶ cells/ml in RPMI 1640 medium containing 2 mMglutamine, 1×non-essential amino acids, 1 mM sodium pyruvate, 100 μg/mlkanamycin, 5×10⁻⁵ M 2-mercaptoethanol, and 5% human serum in thepresence of 400 U/ml hrIL-2).

[0209] Cell migration was assessed in 48-well chambers (Neuro Probe,Cabin John, Md., USA) using polyvinylpyrrolidone-free polycarbonatemembranes (Nucleopore) with 5-μm pores for IP-10/MigR transfected cells(Loetscher, P. et al., FEBS Lett. 341: 187-192 (1994)) or with 3-μmpores for human PBL (Loetscher, P. et al., FASEB J., 8: 1055-1060(1994)) RPMI 1640 supplemented with 20 mM Hepes, pH 7.4, and 1%pasteurized plasma protein solution (Swiss Red Cross Laboratory, Bern,Switzerland) was used to dissolve the chemokines (lower wells), and todilute the cells (100,000 receptor transfectants or PBL in the upperwell). After 60 minutes at 37° C., the membrane was removed, washed onthe upper side with PBS, fixed and stained. All assays were done intriplicate, and the migrated cells were counted in five randomlyselected fields at 1,000-fold magnification. Spontaneous migration wasdetermined in the absence of chemoattractant.

[0210] Results—Transfected Cells

[0211] Transfected cells expressing the IP-10/MigR readily migratedtoward IP-10 or Mig, while the non-transfected, parental cells did notrespond (FIG. 3C). Both agonists showed a typically biphasicconcentration dependence IP-10 induced migration at concentrations above1 nM, whereas the response of Mig became detectable above 10 nM. Theefficacy, which is measured by the maximum number of migrating cells,was about twice as high for Mig as for IP-10. These results demonstratethat the IP-10/MigR, like all known chemokine receptors in leukocytes,mediates chemotaxis in response to ligand.

[0212] Results—Human Blood Leukocytes

[0213] In agreement with the cellular distribution of the IP-10/MigR,activated human T lymphocytes were found to be highly responsive toIP-10 and Mig (FIGS. 4A-4B). The activity of IP-10 and Mig as inducersof [Ca²⁺]_(i) changes (FIG. 4A) and in vitro chemotaxis (FIG. 4B) wasconsistent with the effects observed using transfected cells expressingthe IP-10/MigR, with IP-10 being more potent but less efficacious thanMig. Activation of the T lymphocytes by culturing in the presence ofIL-2 was required for induction of calcium flux and chemotaxis, and noresponse was observed with freshly isolated blood lymphocytes under theconditions used.

[0214] Materials and Methods for Examples 3-9

[0215] The following materials and methods were used in Examples 3-9.

[0216] Chemokines

[0217] Recombinant human chemokines were obtained from Peprotech (RockyHill, N.J.), except for eotaxin, described previously (Ponath, P. D., etal., J. Clin. Invest., 97:604-612 (1996); see also Ponath et al., WO97/00960, published Jan. 9, 1997), which was a gift from Dr. IanClark-Lewis. ¹²⁵I-labeled chemokines were obtained from Du Pont NEN(Boston, MA).

[0218] Cells and Cell Lines

[0219] Neutrophils and PBMCs were isolated as described (Ponath, P. D.,et al., J. Clin. Invest., 97:604-612 (1996)). To generate CD3 blasts,2×10⁶ PBMC/ml in RPMI-1640 plus 10% FCS were added to tissue cultureplates which had been coated with the anti-CD3 antibody TR66. After 4-6days blasts were removed to fresh media and supplemented with IL-2(kindly provided by Antonio Lanzavecchia, Basel) at 50 units/ml.

[0220] Other cell lines used included transfectants of the L1.2 murinepre B cell lymphoma, expressing either CXCR3 (see below), IL-8 RA(Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)), IL-8 RB(Ponath, P. D., et al., J. Exp. Med., 183:2437-2448 (1996)), CCR2b (G.LaRosa, unpublished), CCR4, CCR5 (Wu, L., et al., Nature, 384:179-183(1996)), or CCR1 (Campbell, J. J., et al., J. Cell Biol., 134:255-266(1996)).

[0221] Preparation of CXCR3 Transfectants

[0222] Cells

[0223] L1.2 cells were grown in RPMI medium 1640, 10% Fetal Clone (fromHyclone, Inc.), 50 U/ml Penicillin/Streptomycin, 1×L-Glutamine, 1 mMNaPyruvate, and 5-5×10⁻⁵M β-Mercaptoethanol. Media components werepurchased from GibcoBRL, except for 10% Fetal Clone, which was purchasedfrom Hyclone, Inc. Two days prior to transfection, the L1.2 cells werediluted 1:5 into fresh medium. This resulted in 150 million cells in logphase growth at a concentration of about 1-3 million cells/ml.

[0224] CXCR3 DNA and Transfection

[0225]E. coli XL1Blue cells (Stratagene, Inc. (Cat# 200236)) weretransformed with pcDNA3-Clone8 (Example 2; Loetscher, M., et al., J.Exp. Med., 784:963-969 (1996)) according to the manufacturer's protocol.Transformants were grown at 37° C. while shaking at 250 rpm in 500 ml ofLB containing 100 μg/ml Ampicillin. The culture was then collected bycentrifugation at 8,000×g, and the plasmid was purified using a Maxiplasmid purification column and protocol (Qiagen, Cat# 12162). Plasmidconcentration and purity were determined using a 1% agrose gel andOD260/280 ratios. Plasmid DNA was suspended in ddH₂O, and stored at −20°C. until use.

[0226] ScaI endonuclease was used to linearize the vector. 100 μg of DNAwas digested with 10 μl of ScaI for 8 hours at 37° C. following themanufacturer's protocol (GibcoBRL, Cat# 15436-017). 20 μg was useddirectly in stable transfection construction. 80 μg was cleaned ofproteins and salts with a Phenol:Chloroform:IsoAmyl Alcohol (25:24:1)extraction, 1000 Ethanol precipitation (with 0.1 volume NH₄COOH), and a700 ethanol wash.

[0227] Stable transfectants of murine pre-B lymphoma cell line (L1.2)were prepared essentially as described (Ponath, P. D., et al., J. Exp.Med., 183:2437-2448 (1996)). 25 million L1.2 cells in 0.8 ml of 1×PBSwere electroporated with 20 μg of linearized DNA, 20 μg linearized DNAthat was then cleaned (see above under linearization of DNA), or withoutDNA. Before electroporation, the L1.2 cells and the DNA were incubatedfor 10 minutes in 50 ml conical tubes (Falcon Model 2070) with gentlemixing (swirling) every 2 minutes. The L1.2 cell-DNA mixture wastransferred into Gene Pulser cuvettes (BioRad, Cat# 165-2088) with a 0.4cm electrode gap. The mixture was then electroporated at 250V and 960μF, with the duration of shock and the actual voltage being measured.After electroporation, the cuvette was left undisturbed for 10 minutesat room temperature. All of the L1.2 cells-DNA mixture was thentransferred to a T-25 flask (Costar), and grown for two days in 10 mlnon-selective medium.

[0228] Selection

[0229] L1.2 cells expressing CXCR3 were then subjected to selection forneomycin resistance. After two days of growth in non-selective medium,10 ml of 1.6 g/L Geneticin (GibcoBRL) was added for a finalconcentration of 0.8 g/L (The selective and maintenance concentration).This was then allowed to grow for 10 to 15 days, with fresh selectivemedium added when cells started to over-grow. Fresh selective mediumconsisted of RPMI-1640 supplemented with 10% bovine serum and 800 μg/mlG418.

[0230] The cell surface expression of CXCR3 was assessed by chemotaxis,and ligand binding and Scatchard analysis was also used to monitorsurface expression. After G418 selection, CXCR3 expressing L1.2 cellswere selected based on chemotaxis ability. For each electroporationreaction, 30 ml (800,000 cells/ml) were collected, and suspended in 600μl selective medium. Selective medium, 600 μl, containing 10 nM IP-10,was placed into the bottom chamber of BioCoat cell culture plates fromBecton-Dickinson (Cat# 40575). 100 μl/well of the L1.2 cells were addedinto the top chamber of the BioCoat plates. These cells were then leftto chemotax overnight in a C0₂ incubator at 37° C. The top chambers withthe non-chemotaxing cells were removed. The chemotaxed cells werecollected, transferred into fresh medium, and allowed to grow in a 24well plate. They were subsequently expanded into a T-25 and then a T-75flask from Costar.

[0231] Transfectants expressing high level of receptors were cloned bylimiting dilution. CXCR3 transfected cells were diluted to between 30-3cells/ml in selection medium containing G418. Aliquots were added to96-well tissue culture plates at 100 μl/well. After 14 days at 37° C.and 5% CO₂, wells containing single colonies were identified under aninverted microscope. 50 μl of the cells were then transferred andstained with anti-CXCR3 mAb as above and analyzed by flow cytometry. Thelevel of receptor expression correlated with mean fluorescence intensityand high expressors were selected. Once a stable cell line wasestablished, the line was expanded for use.

[0232] In addition to selection for antibiotic resistance andchemotaxis, CXCR3 transfectant cells can be further selected by sortingfor higher receptor expression by antibody staining, although this wasnot done here. For staining, transfectant cells can be resuspended at5×10⁶/ml in sterile PBS containing 1% bovine serum albumin. Isolated,sterile anti-CXCR3 mAb can be added to a final concentration of 3 μg/mland cells incubated on ice for 30 minutes. After washing with coldsterile PBS, the bound mAb can be detected with FITC-conjugatedanti-mouse IgG, sterilized by filtration through a 0.2 μm filter. Thecells can be washed again and sorted by flow cytometry. The top 5%positive cells can be collected and returned to tissue culture forexpansion under selective conditions (e.g., RPMI-1640 supplemented with10% bovine serum and 800 μg/ml G418).

EXAMPLE 3 IP-10 Binds with High Affinity to a Receptor Expressed on L1.2Cells Transfected with CXCR3 DNA and on Activated T Cells

[0233] Additional binding studies were performed using radiolabeledIP-10. Chemokine binding to target cells was carried out as describedpreviously (Ponath, P. D., et al., J. Clin. Invest., 97:604-612 (1996);Van Riper, G., et al., J. Exp. Med., 177(3):851-856 (1993)). Cells werewashed once in PBS and resuspended in binding buffer (50 mM HEPES, pH7.5, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% BSA, and 0.05% azide) at aconcentration of 10⁷/ml. Aliquots of 50 μl (5×10⁵ cells) were dispensedinto microfuge tubes, followed by the addition of cold competitor(unlabeled IP-10) and radiolabeled chemokine (0.05 nM ¹²⁵I-labeledIP-10). The final reaction volume was 200 μl. Nonspecific binding wasdetermined by incubating cells with radiolabeled chemokines in thepresence of 250-500 nM of unlabeled chemokines. After a 60-minuteincubation at room temperature, the cells were washed three times with 1ml of binding buffer containing 0.5 M NaCl. Cell pellets were thencounted. The competition was presented as the percent specific bindingas calculated by 100×[(S−B)/(T−B)], where S is the radioactivity of thesample, B is background binding, and T is total binding withoutcompetitors. Background binding was obtained by incubating cells withradiolabeled chemokine and at least 400-fold excess of unlabeledchemokines. Duplicates were used throughout the experiments and thestandard deviations were always <10% of the mean. All experiments wererepeated at least three times. Curve fit and concentrations that inhibit50% specific binding (IC₅₀) were calculated by KaleidaGraph software(Synergy Software, Reading, Pa.).

[0234] FIGS. 5A-5B show that ¹²⁵I-labeled IP-10 bound to L1.2 cellstransfected with CXCR3 (FIG. 5A) and to CD3-activated T cells (FIG. 5B),and that this binding could be inhibited with increasing concentrationsof cold IP-10. Scatchard analysis revealed that IP-10 bound to L1.2CXCR3 transfectants with a Kd of 614 pM, and that these transfectantsexpressed 37,000 receptors per cell (FIG. 5A, inset). A similar analysisof anti-CD3 activated, IL-2 stimulated T cells revealed a Kd of 156 pM,and 17,000 receptors per cell (FIG. 5B). ¹²⁵I-labeled IP-10 binding toactivated T cells could be totally inhibited by cold Mig under the sameconditions, although Mig was slightly less efficient at blocking¹²⁵I-labeled IP-10 binding than was cold IP-10 (not shown). IP-10 andMig bind CXCR3 with high affinity (Kd ˜150-600 pM).

EXAMPLE 4 Production and Characterization of Monoclonal Antibodies(mAbs) Specific for CXCR3

[0235] To develop antagonists of CXCR3, and to study receptor expressionand regulation, a panel of mAbs was produced by immunizing mice with asynthetic peptide corresponding to the N-terminus of this receptor.These mAbs specifically recognized CXCR3 transfectants, but not a rangeof other receptor transfectants.

[0236] mAb Production and Flow Cytometry

[0237] mAbs reactive with CXCR3 were generated by immunizing Balb/C micewith 10 μg of 37-mer synthetic peptide corresponding to the first 37N-terminal amino acids of CXCR3 (see also, Loetscher M., et al., J. Exp.Med., 184:963-969 (1996)), five times over a period of 10 weeks. Thispeptide was synthesized and coupled to purified protein derivative oftuberculin (Severn Biotech Ltd., Kidderminster, U.K.). The firstimmunization was intraperitoneal (IP) with Freund's Complete Adjuvant(FCA). The second, third and fourth immunizations were IP with Freund'sIncomplete Adjuvant (FIA), and the final immunization was with peptideconjugate alone (no adjuvant), and was administered intravenously (IV).Four days after the last immunization, the spleen was taken and cellfusion performed using the cell line SP2/0, as described (Coligan, J.E., et al., Current Protocols In Immunology (John Wiley and Sons, NewYork), Unit 2.5.4 (1992)). mAbs were generated that reacted with theN-terminal 37-mer peptide as assessed by ELISA (Coligan, J. E., et al.,Current Protocols In Immunology (John Wiley and Sons, New York), Unit2.1.3 (1992)). mAbs reactive with CXCR3 were identified usinguntransfected and CXCR3 transfected L1.2 cells or 300.19 cells (a murineB cell line, Loetscher, M. et al., J. Exp. Med., 184: 963-969, (1996)),and immunofluorescent staining and analysis using a FACScan® (BectonDickinson & Co., Mountain View, Calif.).

[0238] Monoclonal antibodies specific for CXCR3 were generated. EightmAbs were found to recognize surface expressed CXCR3, as judged bystaining of CXCR3 transfected L1.2 cells, but not untransfected L1.2cells or L1.2 cells transfected with other receptor types. The FACSprofile of one of these mAbs, 1C6, is shown in FIG. 6. These mAbs alsostained human T cells and T cell clones that had been activated in vitrowith PHA or anti-CD3 (illustrated in FIG. 7C with 1C6). However, theseanti-CXCR3 mAbs were unreactive with neutrophils (illustrated in FIG. 7Awith 1C6), monocytes, or eosinophils (not shown). This pattern ofreactivity was consistent with the analysis by Northern blot. However,the phenotypic analysis unexpectedly revealed the expression of CXCR3 ona large subset of circulating lymphocytes (FIG. 7B). This expressionpattern was observed in all individuals examined, indicating that CXCR3is normally expressed on a subset of blood lymphocytes. CXCR3 was foundto mark a population of circulating T cells, which were contained withinthe CD45RO+ (memory) subset.

[0239] Murine hybridoma 1C6 (also referred to as LS77-1C6) was depositedon Mar. 28, 1997 at the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., 20852, in accordance with the terms of theBudapest Treaty, under Accession Number ______.

EXAMPLE 5 CXCR3 is Expressed on Activated/Memory T Cells

[0240] Flow Cytometry

[0241] mAbs to CXCR1, CXCR2, CXCR3, and CCR5 have been described (Qin,S., et al., Eur. J. Immunol., 26:640-647 (1996); Heath, H., et al., J.Clin. Invest., in press (1997)). Anti-CXCR4 mAb 12G5 (Endres, M. J., etal., Cell, 87:745-756 (1996)) was kindly provided by Jim Hoxie (Univ.Penn.). PE-conjugated nAbs to CD4, CD8, CD14, CD20, CD25, CD26, CD69,CD45RO, CD45RA, CD95, and anti-CD3 and anti-CD4 Cy-Chrome were suppliedby PharMingen (La Jolla, Calif.).

[0242] To assess reactivity of mAbs against transfected cells orleukocytes, indirect immunofluorescence and flow cytometry were used.Cells were washed once with PBS, and resuspended in 100 μl PBScontaining 2% human serum and 0.1% sodium azide (staining buffer), 5μg/ml purified antibody, 5 μg/ml IgG_(2a) isotype matched control mAb(Sigma Chemical Co., St. Louis, MO) or 50 μl hybridoma culturesupernatant. After 20 min at 4° C., cells were washed twice withstaining buffer, and resuspended in 50 μl FITC-conjugated affinitypurified F(ab′)₂ goat anti-mouse IgG (Jackson ImmunoResearchLaboratories). After incubating for 20 min at 4° C., cells were washedtwice in staining buffer and analyzed on the FACScan® to determine thelevel of surface expression. Propidium iodide was used to exclude deadcells.

[0243] A two color immunofluorescence analysis of lymphocytes showedthat it was mostly CD3+ cells that expressed CXCR3, although a smallproportion of CD20+ (B) cells and CD56+ (NK) cells also expressed thisreceptor (FIG. 8). A three color analysis of T cells, performed usinganti-CD3 Cy-Chrome to label T cells, showed that a portion of the CD4+cells and a portion of the CD8+ cells expressed CXCR3 (FIG. 9). Ananalysis using markers of cellular activation, such as CD25 and CD69,revealed that activated T cells generally expressed this receptor.CXCR3+ T cells were CD95+, CD45RO+, and CD45RA^(low), a phenotypeconsistent with previous activation. The expression of CXCR3 and CCR5, achemokine receptor that is also biased in its expression to previouslyactivated T cells (Wu, L., personal communication), was also compared.FIG. 9 shows that the CCR5+ cells in blood were contained within theCXCR3+ subset, and that CXCR3 was more widely expressed that CCR5.Unlike other T cell chemokine receptors, such as CCR5 or CXCR4, CXCR3was expressed on the majority of circulating, activated T cells.

EXAMPLE 6 Anti-CXCR3 mAb Blocks IP-10 Binding and Chemotaxis

[0244] Chemotaxis of human leukocytes was assessed using a modificationof a transendothelial assay (Carr, M. W., et al., Proc. Natl. Acad. Sci.USA, 91(9):3652-3656 (1994)), which has been described previously(Ponath, P. D., et al., J. Clin. Invest., 97:604-612 (1996)), using theECV 304 endothelial cell line (European Collection of Animal CellCultures, Porton Down, Salisbury, U.K.). Cells that had migrated to thebottom chamber were placed in a tube, and relative cell counts wereobtained using the FACScan®.

[0245] The anti-peptide mAbs were also tested for their ability toinhibit the chemotaxis of CD3 activated T cell blasts. The results formAbs designated 1A5, 1C6, 3AB, 5F10, 10C6 and 10G12 are illustrated inFIG. 10. One mAb, 1C6, was superior to the other mAbs in its ability toblock the chemotaxis of T cells to IP-10. mAb 1C6 was able to inhibitcompletely the chemotaxis of T cells to IP-10 in a dose-dependentmanner, with an IC₅₀ of ˜0.8 μg/ml (FIG. 11). A concentration of 2-5μg/ml was achieved 100% inhibition, using an optimal concentration ofIP-10 (12.5 nM) in the bottom of the transwell. 1C6 was unable tosignificantly inhibit T cell chemotaxis to MCP-1 under the conditionsused (FIG. 10), which occurs through the chemokine receptor CCR2b.

[0246] mAb 1C6 was also able to block completely ¹²⁵I-labeled IP-10binding to activated T cells, with an IC₅₀ of 0.16 μg/ml (FIG. 12).Between 1 and 10 μg/ml of antibody gave complete inhibition. Thecomplete inhibition of both IP-10 binding and chemotaxis by mAb 1C6indicates that activated T cells do not express another receptor thatbinds this chemokine.

[0247] Northern blot analysis indicated that CXCR3 was expressed inactivated T cells. Using a panel of specific mAbs, CXCR3 was found to beexpressed on a subset of blood T cells, as well as on other leukocytetypes (B cells and NK cells). CXCR3 expressing T cells had a phenotypeconsistent with previous activation, i.e. CD45RO+, CD26+ (Example 5).Staining of T cells was markedly increased when T cells were activatedby CD3 and IL-2, which correlated with increased cell migration inresponse to IP-10 and readiolabeled ligand binding.

[0248] The poor responsiveness of blood T cells to IP-10 or Mig, atleast in chemotaxis assays, appears anomalous. A possible explanation isthat factors other than receptor expression may determine cellularresponsiveness to chemoattractants. Appropriate G protein coupling maybe necessary for signalling. Another possibility is that the bloodseparation procedure disrupts this receptor, as has been observed forIL-8 receptors on T cells and NK cells (C. R. Mackay). Injection ofIP-10 into the skin of in appropriate experimental animal can addressthe significance of CXCR3 expression on blood T cells. On activated Tcells, IP-10 is one of the most potent chemoattractants. Activation of Tcells may induce the receptor signalling molecules or coupling neededfor signal transduction.

EXAMPLE 7 mAb 1C6 Selectively Inhibits [Ca²⁺]_(i) by T cells in Responseto IP-10, but Not Mig

[0249] Intracellular calcium concentration ([Ca²⁺]i) was determined asfollows. A stock solution of Fura-2 AM (Molecular Probes, Eugene, Oreg.)was prepared by dissolving 50 μg of the dye in 44 μl of DMSO.Immediately prior to addition to cells, this stock was diluted 1:100into HBSS with Ca²+ and Mg²+ and 2% BSA. Fura-2 AM was added to cells ata final concentration of 0.2 moles/10⁶ cells at 37° C. for 30 minutes.Following labeling, excess dye was removed by centrifugation and cellswere resuspended at a concentration of 10⁶/ml in 125 mM NaCl, 5 mM KCl,1 nM MgCl₂, 1 mM CaCl₂, 0.5 mM glucose, 0.025% BSA and 20 nM HEPES, pH7.4. [Ca²+]_(i) was measured using excitation at 340 and 380 nm on aHitachi F-2000 fluorescence spectrometer. Calibration was performedusing 1% NP-40 for total release and 25 μM EGTA to chelate free Ca²⁺.

[0250] IP-10 and Mig induce [Ca²⁺]_(i) by human T cells, and eachchemokine was able to completely desensitize responses to the otherchemokine (FIGS. 13A-13B). A titration of the chemokines revealed thatmaximal [Ca²⁺]_(i) was achieved with as little as 2 nM IP-10 or 2 nMMig. To examine the agonist/antagonist function of mAb 1C6, activated Tcells were assessed for [Ca²⁺]_(i) following injection of mAb 1C6, or anirrelevant isotype-matched control mAb. T cells injected with anirrelevant isotype-matched control 1C6 showed a robust [Ca²⁺]_(i)response to subsequent injection of IP-10 (not shown). However, T cellstreated with mAb 1C6 showed no [Ca²⁺]_(i) upon stimulation with 2 nMIP-10. [Ca²⁺]_(i) in activated T cells in response to IP-10 wascompletely suppressed by 12.5 mg of 1C6. However, as much as 50 μg/ml of1C6 had no effect on the response of T cells to 2 nM Mig (FIG. 13D),indicating that the two ligands were differentially affected by thismAb. As a control, mAb 1C6 was tested for its effects on the [Ca²⁺]_(i)of T cells in response to MIP-1α or RANTES, which occurs throughreceptors other than CXCR3 (not shown). The mAb had no effect under theconditions used. mAb 1C6, which inhibits IP-10 binding and IP-10-inducedchemotaxis, also inhibited calcium flux by activated T cells, but didnot inhibit Mig-induced calcium flux under these conditions. Theseresults suggest the IP-10 and Mig bind and/or signal through differentregions of CXCR3, and that mAb 1CG was able to block the IP-10 bindingsite and subsequent signalling. Thus, it is possible to develop receptorantagonists which selectively inhibit the effects of individualchemokines, illustrated here using mAb 1C6.

[0251] As discussed below, staining of CXCR3 transfectants can beinhibited by a peptide comprising the first 15 N-terminal amino acidresidues of CXCR3. The inhibition of IP-10 binding and subsequentresponses by mAb 1C6 suggests that this region is of particularfunctional importance for ligand binding. Based upon the inability of1C6 to inhibit Mig-induced calcium flux under the conditions used, theepitope recognized by mAb 1C6, which appears to be important for IP-10binding and signalling, does not appear to be involved in Mig bindingand/or signalling.

EXAMPLE 8 Epitope Mapping

[0252] Peptides were ordered from Genemed, South San Franciso, Calif.The peptides, each 15 amino acids long, correspond to different portionsof the first 45 N-terminal residues of CXCR3 protein:

[0253] P1: MVLEVSDHQVLNDAE (SEQ ID NO:2, residues 1-15)

[0254] P2: VAALLENFSSSYDYG (SEQ ID NO:2, residues 16-30)

[0255] P3: ENESDSCCTSPPCPQ (SEQ ID NO:2, residues 31-45)

[0256] The peptides were first dissolved in DMSO and diluted to 1 mg/mlin phosphate buffered saline (PBS). To test tie ability of the peptidesto block staining with 1C6 antibody, 1/g/ml of purified 1C6 antibody wasincubated in 1×PBS, 5% Fetal Calf Serum (FCS) with CXCR3 L1.2transfectants (10⁵ cells; see Materials and Methods for Examples 3-9) inthe presence of 100 μg/ml of each peptide for 30 minutes at 4° C. Finalvolume was 100 μl. Positive staining was carried out in the absence ofpeptide. Bound mAb was detected with anti-mouse IgG-FITC and the resultswere analyzed by flow cytometry.

[0257] Staining with mAb 1C6 was completely inhibited by PI, suggestingthat the mAb recognizes an epitope in the first N-terminal 15 aminoacids of the CXCR3 protein (FIGS. 14A-14D). Binding of another mAbdesignated 3A8 (also referred to as LS77-3A8), which was produced fromthe same fusion (LS-77) as mAb 1C6, was also inhibited by the PIpeptide.

EXAMPLE 9 Production of Anti-CXCR3 Monoclonal Antibodies by Immunizationwith Transfected Cells

[0258] Additional anti-CXCR3 monoclonal antibodies were generated byimmunization of mice with L1.2 cells transfected with a CXCR3 constructand expressing high levels of human CXCR3 (see Materials and Methodsabove). Immunization and generation of fusion hybridomas was performedas described (Qin, S. et al., Eur. J. Immunol., 26: 640 (1996); andHeath, H. et al., J. Clin. Invest., 99(2): 178 (1997)). Anti-CXCR3 mAbswere identified by positive staining of activated human T cells andCXCR3 transfectants. Eight anti-CXCR3 antibodies were obtained in onefusion (LS-104).

[0259] The ability of these antibodies to inhibit binding of IP-10 toCXCR3 was tested. Tissue culture supernatants (25 μl) from the positiveclones were incubated with CXCR3 L1.2 transfectant cells (Materials andMethods for Examples 3-9) and 0.05 nM of radiolabeled ¹²⁵I-labeled IP-10in 1×PBS, 5S Fetal Calf Serum (FCS) (100 μl final volume) for 30 minutesat 4° C. Supernatants of mAb 1C6 and an anti-CXCR2 (IL-8 receptor B) mAbwere also used as specific and non-specific mAb controls, respectively.Total binding was determined in the absence of antibodies. Backgoundbinding was obtained using 40 nM unlabeled IP-10 as the competitor andthis value was used to calculate the percentage of inhibition by themAbs. The results are shown in FIG. 15. All antibodies from this fusion(LS-104) were able to block IP-10 binding, with the percentageinhibition ranging from 50-70a.

[0260] These mAbs were also assessed for their ability to inhibitsignalling (ability to induce Ca²⁺ flux) essentially as described inExample 7, but none of the antibodies was able to block Mig-mediatedCa²⁺ signalling under the conditions used. Antibody inhibition ofIP-10-mediated CXCR3 binding and signalling, but not Mig-mediatedsignalling via CXCR3, indicates that these antibodies can selectivelyinhibit CXCR3 functions mediated by IP-10.

[0261] Epitope mapping studies were also carried out using theantibodies from the LS-104 fusion essentially as described in Example 8.The results indicate that a variety of binding sites are recognized(Table 2). The results suggest that three of the antibodies recognizeepitope(s) within the first 15 N-terminal amino acids of CXCR3, and twoof the antibodies recognize epitope(s) within amino acids 16-30 ofCXCR3. P3 peptide did not block staining with any of these antibodies,indicating that none of these antibodies bound the peptide representingamino acids 31-45 of CXCR3. Staining using the three remaining mAbscould not be significantly inhibited by any of the peptides under theconditions used, suggesting that these mAbs my bind epitopes comprisingoverlapping segments of the peptides, conformational epitopes displayedon the cell surface or epitopes on other parts of the receptor. Thesedata also suggest that mAbs against various portions of CXCR3 can beobtained by immunizing mice with receptor transfectants. TABLE 2 Epitopemapping of anti-CXCR3 mAbs Fusion Number Peptides which inhibit mAbBinding mAb Name staining of CXCR3 transfectants Region LS-77¹ 1C6 P1 AA1-15 3A8 P1 AA 1-15 LS-104² 2F8 none 3A12 P1 AA 1-15 3E2 P1 AA 1-15 4B4P2 AA 16-30 4D2 none 5B12 none 7B8 P2 AA 16-30 8D5 P1 AA 1-15

EXAMPLE 10 Immunohistochemical Analysis of Normal and Inflamed TissuesUsing mAb 1C6

[0262] Tissues

[0263] Human tissues (normal and inflamed) were obtained from theNational Disease Research Institute, a service organization funded bythe National Institutes of Health. Normal macaque (Macaca mulatta)tissues were obtained from the New England Regional Primate ResearchCenter, Southboro, Mass.

[0264] Immunohistochemistry

[0265] Alkaline Phosphatase Technique. Tissue was sectioned at athickness of 4 μm, desiccated, and then fixed in 2%paraformaldehyde/0.5×PBS for 10 minutes at 40C. After PBS washing,nonspecific antibody binding sites were blocked with 10% normal goatserum/5% human AB serum/PBS for 30 minutes at room temperature. Next,the purified, anti-CXCR3 murine mob 1C6, was diluted to a concentrationof 10 μg/ml in 0.3% Triton X 100/0.2% Tween 20/1% FCS/5% human AB serum,and 0.1% sodium azide, and applied to tissue sections which wereincubated overnight at 4° C. An isotype-matched irrelevant monoclonalantibody was used as a negative control on step sections of tissues(IgG₁, MOPC-21, Sigma, St. Louis, Mo.). Subsequently, biotinylated goatanti-mouse IgG (Vector, Burlingame, Calif.) and avidin-biotin-alkalinephosphatase complexes (Biogenex, San Ramon, Calif.) were added insequence. Fast Red (Biogenex, San Ramon, Calif.), containing levamisolto block endogenous alkaline phosphatase activity, was used as thechromogen and Mayers hematoxylin as the counterstain.

[0266] Results

[0267] Human and macaque normal lymph node: In both species, stainingwas limited to 70-80% of lymphocytes within the paracortex and medullarycords, consistent with CXCR3 expression on T lymphocytes.

[0268] Human and macaque spleen: In both species, staining was limitedto lymphocytes along the periphery of lymphoid follicles of white pulpand scattered lymphocytes within splenic sinusoids. This pattern isconsistent with CXCR3 expression on T lymphocytes.

[0269] Human thymus: The thymic medulla contained scattered CXCR3immunoreactive mononuclear cells morphologically consistent withlymphocytes.

[0270] This analysis revealed that macaque CXCR3 is recognized by mAb1C6. Separate studies showed that CXCR3 is upregulated by culturingmacaque cells T lymphocytes with concanavalin A and IL-2, macaque T cellblasts can chemotax in response to human IP-10, mAb 1C6 can block thischemotaxis, and prior incubation with human IP-10 desensitizes macaqueblasts as assessed by chemotaxis.

[0271] In the following discussion, variable numbers of CXCR3immunoreactive “mononuclear cells” were identified in both normal andinflamed tissues. These mononuclear cells are most likely T lymphocytesfor the following reasons: a) flow cytometry revealed that the majorityof CXCR3⁺ cells are T lymphocytes, although CXCR3 has been detected onsome B cells and NK cells, but not on other mononuclear cells; b) withinthe lymph node and spleen, lymphocytes in regions known to be populatedby T cells are the only cells that are immunoreactive for CXCR3; and c)the CXCR3 immunoreactive mononuclear cells in the tissues listed beloware morphologically consistent with lymphocytes.

[0272] Non-inflamed human tissues: In non-inflamed human tissuesincluding heart, liver, kidney, lung, skin, breast, skeletal muscle,salivary gland, pancreas, vagina, uterus, and ovary, CXCR3 expressionwas limited to rare, scattered interstitial mononuclear cells. Insections of small and large intestine, mononuclear cells expressingCXCR3 were observed within the lamina propria and Peyer's patches. Inliver, periductal lymphocytes were also stained and hepatocytes werelightly stained.

[0273] Human brain: No staining was observed.

[0274] Inflamed human tissues: Several sections of chronically inflamedtissue characterized by interstitial and perivascular accumulation ofmononuclear cells were examined for CXCR3 expression. In these tissues,including tissue from human patients with interstitial nephritis (1case, kidney tissue), ulcerative colitis (1 case, colon tissue),enteritis (1 case, small intestine) and chronic vaginitis (4 cases,vagina), about 50%-90% of mononuclear cells were immunoreactive forCXCR3. Thus, compared to normal tissues, chronically inflamed tissuescontained a greater number of interstitial mononuclear cells, and agreater percentage of these cells were immunoreactive for CXCR3.

[0275] Analysis of inflamed tissues revealed that about 50-90% oflymphocytes expressed CXCR3, whereas much lower percentages oflymphocytes in the corresponding normal tissue expressed CXCR3. Theseobservations suggest specific recruitment of lymphocytes expressingCXCR3, most likely T lymphocytes expressing CXCR3, to sites of chronicinflammation. CXCR3 appears to mark T cells with a predilection forhoming or migration to inflammatory sites.

[0276] Equivalents

[0277] Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1 4 1670 base pairs nucleic acid double unknown CDS 69..1172 1CCAACCACAA GCACCAAAGC AGAGGGGCAG GCAGCACACC ACCCAGCAGC CAGAGCACCA 60GCCCAGCC ATG GTC CTT GAG GTG AGT GAC CAC CAA GTG CTA AAT GAC GCC 110 MetVal Leu Glu Val Ser Asp His Gln Val Leu Asn Asp Ala 1 5 10 GAG GTT GCCGCC CTC CTG GAG AAC TTC AGC TCT TCC TAT GAC TAT GGA 158 Glu Val Ala AlaLeu Leu Glu Asn Phe Ser Ser Ser Tyr Asp Tyr Gly 15 20 25 30 GAA AAC GAGAGT GAC TCG TGC TGT ACC TCC CCG CCC TGC CCA CAG GAC 206 Glu Asn Glu SerAsp Ser Cys Cys Thr Ser Pro Pro Cys Pro Gln Asp 35 40 45 TTC AGC CTG AACTTC GAC CGG GCC TTC CTG CCA GCC CTC TAC AGC CTC 254 Phe Ser Leu Asn PheAsp Arg Ala Phe Leu Pro Ala Leu Tyr Ser Leu 50 55 60 CTC TTT CTG CTG GGGCTG CTG GGC AAC GGC GCG GTG GCA GCC GTG CTG 302 Leu Phe Leu Leu Gly LeuLeu Gly Asn Gly Ala Val Ala Ala Val Leu 65 70 75 CTG AGC CGG CGG ACA GCCCTG AGC AGC ACC GAC ACC TTC CTG CTC CAC 350 Leu Ser Arg Arg Thr Ala LeuSer Ser Thr Asp Thr Phe Leu Leu His 80 85 90 CTA GCT GTA GCA GAC ACG CTGCTG GTG CTG ACA CTG CCG CTC TGG GCA 398 Leu Ala Val Ala Asp Thr Leu LeuVal Leu Thr Leu Pro Leu Trp Ala 95 100 105 110 GTG GAC GCT GCC GTC CAGTGG GTC TTT GGC TCT GGC CTC TGC AAA GTG 446 Val Asp Ala Ala Val Gln TrpVal Phe Gly Ser Gly Leu Cys Lys Val 115 120 125 GCA GGT GCC CTC TTC AACATC AAC TTC TAC GCA GGA GCC CTC CTG CTG 494 Ala Gly Ala Leu Phe Asn IleAsn Phe Tyr Ala Gly Ala Leu Leu Leu 130 135 140 GCC TGC ATC AGC TTT GACCGC TAC CTG AAC ATA GTT CAT GCC ACC CAG 542 Ala Cys Ile Ser Phe Asp ArgTyr Leu Asn Ile Val His Ala Thr Gln 145 150 155 CTC TAC CGC CGG GGG CCCCCG GCC CGC GTG ACC CTC ACC TGC CTG GCT 590 Leu Tyr Arg Arg Gly Pro ProAla Arg Val Thr Leu Thr Cys Leu Ala 160 165 170 GTC TGG GGG CTC TGC CTGCTT TTC GCC CTC CCA GAC TTC ATC TTC CTG 638 Val Trp Gly Leu Cys Leu LeuPhe Ala Leu Pro Asp Phe Ile Phe Leu 175 180 185 190 TCG GCC CAC CAC GACGAG CGC CTC AAC GCC ACC CAC TGC CAA TAC AAC 686 Ser Ala His His Asp GluArg Leu Asn Ala Thr His Cys Gln Tyr Asn 195 200 205 TTC CCA CAG GTG GGCCGC ACG GCT CTG CGG GTG CTG CAG CTG GTG GCT 734 Phe Pro Gln Val Gly ArgThr Ala Leu Arg Val Leu Gln Leu Val Ala 210 215 220 GGC TTT CTG CTG CCCCTG CTG GTC ATG GCC TAC TGC TAT GCC CAC ATC 782 Gly Phe Leu Leu Pro LeuLeu Val Met Ala Tyr Cys Tyr Ala His Ile 225 230 235 CTG GCC GTG CTG CTGGTT TCC AGG GGC CAG CGG CGC CTG CGG GCC ATG 830 Leu Ala Val Leu Leu ValSer Arg Gly Gln Arg Arg Leu Arg Ala Met 240 245 250 CGG CTG GTG GTG GTGGTC GTG GTG GCC TTT GCC CTC TGC TGG ACC CCC 878 Arg Leu Val Val Val ValVal Val Ala Phe Ala Leu Cys Trp Thr Pro 255 260 265 270 TAT CAC CTG GTGGTG CTG GTG GAC ATC CTC ATG GAC CTG GGC GCT TTG 926 Tyr His Leu Val ValLeu Val Asp Ile Leu Met Asp Leu Gly Ala Leu 275 280 285 GCC CGC AAC TGTGGC CGA GAA AGC AGG GTA GAC GTG GCC AAG TCG GTC 974 Ala Arg Asn Cys GlyArg Glu Ser Arg Val Asp Val Ala Lys Ser Val 290 295 300 ACC TCA GGC CTGGGC TAC ATG CAC TGC TGC CTC AAC CCG CTG CTC TAT 1022 Thr Ser Gly Leu GlyTyr Met His Cys Cys Leu Asn Pro Leu Leu Tyr 305 310 315 GCC TTT GTA GGGGTC AAG TTC CGG GAG CGG ATG TGG ATG CTG CTC TTG 1070 Ala Phe Val Gly ValLys Phe Arg Glu Arg Met Trp Met Leu Leu Leu 320 325 330 CGC CTG GGC TGCCCC AAC CAG AGA GGG CTC CAG AGG CAG CCA TCG TCT 1118 Arg Leu Gly Cys ProAsn Gln Arg Gly Leu Gln Arg Gln Pro Ser Ser 335 340 345 350 TCC CGC CGGGAT TCA TCC TGG TCT GAG ACC TCA GAG GCC TCC TAC TCG 1166 Ser Arg Arg AspSer Ser Trp Ser Glu Thr Ser Glu Ala Ser Tyr Ser 355 360 365 GGC TTGTGAGGCCGGA ATCCGGGCTC CCCTTTCGCC CACAGTCTGA CTTCCCCGCA 1222 Gly LeuTTCCAGGCTC CTCCCTCCCT CTGCCGGCTC TGGCTCTCCC CAATATCCTC GCTCCCGGGA 1282CTCACTGGCA GCCCCAGCAC CACCAGGTCT CCCGGGAAGC CACCCTCCCA GCTCTGAGGA 1342CTGCACCATT GCTGCTCCTT AGCTGCCAAG CCCCATCCTG CCGCCCGAGG TGGCTGCCTG 1402GAGCCCCACT GCCCTTCTCA TTTGGAAACT AAAACTTCAT CTTCCCCAAG TGCGGGGAGT 1462ACAAGGCATG GCGTAGAGGG TGCTGCCCCA TGAAGCCACA GCCCAGGCCT CCAGCTCAGC 1522AGTGACTGTG GCCATGGTCC CCAAGACCTC TATATTTGCT CTTTTATTTT TATGTCTAAA 1582ATCCTGCTTA AAACTTTTCA ATAAACAAGA TCGTCAGGAC CTTTTTTTTT TTTTTTTTTT 1642TTTTTTTTTT TTTTTTTTTT TTTTTTTT 1670 368 amino acids amino acid linearprotein 2 Met Val Leu Glu Val Ser Asp His Gln Val Leu Asn Asp Ala GluVal 1 5 10 15 Ala Ala Leu Leu Glu Asn Phe Ser Ser Ser Tyr Asp Tyr GlyGlu Asn 20 25 30 Glu Ser Asp Ser Cys Cys Thr Ser Pro Pro Cys Pro Gln AspPhe Ser 35 40 45 Leu Asn Phe Asp Arg Ala Phe Leu Pro Ala Leu Tyr Ser LeuLeu Phe 50 55 60 Leu Leu Gly Leu Leu Gly Asn Gly Ala Val Ala Ala Val LeuLeu Ser 65 70 75 80 Arg Arg Thr Ala Leu Ser Ser Thr Asp Thr Phe Leu LeuHis Leu Ala 85 90 95 Val Ala Asp Thr Leu Leu Val Leu Thr Leu Pro Leu TrpAla Val Asp 100 105 110 Ala Ala Val Gln Trp Val Phe Gly Ser Gly Leu CysLys Val Ala Gly 115 120 125 Ala Leu Phe Asn Ile Asn Phe Tyr Ala Gly AlaLeu Leu Leu Ala Cys 130 135 140 Ile Ser Phe Asp Arg Tyr Leu Asn Ile ValHis Ala Thr Gln Leu Tyr 145 150 155 160 Arg Arg Gly Pro Pro Ala Arg ValThr Leu Thr Cys Leu Ala Val Trp 165 170 175 Gly Leu Cys Leu Leu Phe AlaLeu Pro Asp Phe Ile Phe Leu Ser Ala 180 185 190 His His Asp Glu Arg LeuAsn Ala Thr His Cys Gln Tyr Asn Phe Pro 195 200 205 Gln Val Gly Arg ThrAla Leu Arg Val Leu Gln Leu Val Ala Gly Phe 210 215 220 Leu Leu Pro LeuLeu Val Met Ala Tyr Cys Tyr Ala His Ile Leu Ala 225 230 235 240 Val LeuLeu Val Ser Arg Gly Gln Arg Arg Leu Arg Ala Met Arg Leu 245 250 255 ValVal Val Val Val Val Ala Phe Ala Leu Cys Trp Thr Pro Tyr His 260 265 270Leu Val Val Leu Val Asp Ile Leu Met Asp Leu Gly Ala Leu Ala Arg 275 280285 Asn Cys Gly Arg Glu Ser Arg Val Asp Val Ala Lys Ser Val Thr Ser 290295 300 Gly Leu Gly Tyr Met His Cys Cys Leu Asn Pro Leu Leu Tyr Ala Phe305 310 315 320 Val Gly Val Lys Phe Arg Glu Arg Met Trp Met Leu Leu LeuArg Leu 325 330 335 Gly Cys Pro Asn Gln Arg Gly Leu Gln Arg Gln Pro SerSer Ser Arg 340 345 350 Arg Asp Ser Ser Trp Ser Glu Thr Ser Glu Ala SerTyr Ser Gly Leu 355 360 365 28 base pairs nucleic acid single unknownmodified_base 11 /mod_base= i modified_base 12 /mod_base= imodified_base 23 /mod_base= i modified_base 26 /mod_base= i 3 GGGCTGCAGCNNTKKCMGAC MTNCTNYT 28 27 base pairs nucleic acid single unknownmodified_base 10 /mod_base= i modified_base 16 /mod_base= imodified_base 18 /mod_base= i 4 GGGTCTAGAN GGGTTNANRC ARCWRYG 27

We claim:
 1. An isolated nucleic acid encoding a mammalian CXCR3 proteinor functional variant thereof, wherein said protein or variant canselectively bind one or more chemokines and can mediate cellularsignalling and/or a cellular response in response thereto.
 2. Theisolated nucleic acid of claim 1, wherein said nucleic acid canhybridize under moderate stringency conditions to a second nucleic, saidsecond nucleic acid having the sequence of FIG. 1 (SEQ ID NO:1), itscomplement, or a portion of the sequence of FIG. 1 (SEQ ID NO:1) or itscomplement comprising the coding sequence.
 3. The isolated nucleic acidof claim 1, wherein the isolated nucleic acid is essentially pure. 4.The isolated nucleic acid of claim 1, wherein the protein or variant canselectively bind a chemokine selected from the group consisting of humanIP-10, human Mig, a mammalian homolog of human IP-10, a mammalianhomolog of human Mig.
 5. The isolated nucleic acid of claim 1, whereinthe mammalian CXCR3 protein or functional variant thereof is a humanCXCR3 protein or functional variant thereof.
 6. The isolated nucleicacid of claim 1 comprising SEQ ID NO:1, its complement, or a portion ofSEQ ID NO: 1 or its complement comprising the coding sequence.
 7. Theisolated nucleic acid of claim 1, wherein the a human CXCR3 protein hasan amino acid sequence as set forth in SEQ ID NO:2.
 8. An isolatednucleic acid construct comprising a nucleic acid of claim
 1. 9. Theisolated nucleic acid construct of claim 8, wherein the nucleic acid isoperably linked to an expression control sequence.
 10. The isolatednucleic acid construct of claim 8, the nucleic acid comprising SEQ IDNO:1, its complement, or a portion of SEQ ID NO:1 or its complementcomprising the coding sequence.
 11. The isolated nucleic acid constructof claim 8, wherein said nucleic acid encodes a polypeptide having anamino acid sequence as set forth in FIG. 2 (SEQ ID NO:2).
 12. Theisolated nucleic acid construct of claim 8, wherein the nucleic acidencodes a fusion protein comprising a mammalian CXCR3 protein, andoptionally wherein the coding sequence is operably linked to anexpression control sequence.
 13. A host cell comprising a recombinantnucleic acid encoding a mammalian CXCR3 protein or functional variantthereof, wherein said protein can selectively bind one or morechemokines and can mediate cellular signalling and/or a cellularresponse in response thereto.
 14. The host cell of claim 13, wherein thenucleic acid is operably linked to an expression control sequence. 15.The host cell of claim 13, wherein the nucleic acid encodes a humanCXCR3 protein encoded by SEQ ID NO:1.
 16. An isolated mammalian CXCR3protein or functional variant thereof, wherein said protein canselectively bind one or more chemokines and can mediate cellularsignalling and/or a cellular response in response thereto.
 17. Theisolated mammalian CXCR3 protein or functional variant thereof of claim16, wherein the mammal is a human and the protein can selectively bindone or more chemokines selected from the group consisting of human IP-10and human Mig.
 18. The isolated mammalian CXCR3 protein or functionalvariant thereof of claim 16, which is encoded by a nucleic acid whichcan hybridize under moderate stringency conditions to a second nucleicacid, said second nucleic acid having the sequence of FIG. 1 (SEQ IDNO:1), its complement, or a portion of the sequence of FIG. 1 (SEQ IDNO:1) or its complement comprising the coding sequence.
 19. An isolatedhuman CXCR3 protein encoded by the nucleic acid illustrated in FIG. 1(SEQ ID NO:1).
 20. The isolated human CXCR3 protein of claim 19 havingan amino acid sequence as set forth in SEQ ID NO:2.
 21. A fusion proteincomprising a mammalian CXCR3 protein.
 22. A method for producing amammalian CXCR3 protein or variant thereof comprising: (a) introducinginto a host cell a nucleic acid encoding a mammalian CXCR3 protein orvariant thereof, whereby a recombinant host cell is produced having saidcoding sequence operably linked to an expression control sequence; and(b) maintaining the host cells produced in step (a) under conditionswhereby the nucleic acid is expressed.
 23. The method of claim 22,further comprising the step of isolating the mammalian CXCR3 protein orvariant thereof.
 24. A method for producing a mammalian CXCR3 protein orvariant thereof comprising maintaining a host cell containing arecombinant nucleic acid encoding a mammalian CXCR3 protein or variantthereof under conditions suitable for expression of the nucleic acid.25. The method of claim 24 further comprising the step of isolating themammalian CXCR3 protein or variant thereof.
 26. The method of claim 24,wherein the mammal is a human and the protein or variant thereof canselectively bind one or more chemokines selected from the groupconsisting of human IP-10 and human Mig.
 27. An antibody or functionalantibody fragment which binds a mammalian CXCR3 protein.
 28. Theantibody or functional antibody fragment of claim 27, wherein saidantibody or antibody fragment can inhibit one or more functions of amammalian CXCR3 protein.
 29. The antibody or functional antibodyfragment of claim 28, wherein the antibody or fragment inhibits bindingof a ligand to a mammalian CXCR3 protein.
 30. The antibody or functionalantibody fragment of claim 28, wherein said antibody or antibodyfragment can bind a human CXCR3 protein and can inhibit the interactionof a human CXCR3 protein with one or more ligands selected from thegroup consisting of IP-10 and/or Mig.
 31. The antibody or functionalantibody fragment of claim 30, wherein said antibody or fragment cancompete with monoclonal antibody 1C6 for binding to a human CXCR3protein or portion thereof.
 32. The antibody or functional antibodyfragment of claim 28, wherein said antibody or antibody fragmentselectively inhibits the interaction of a human CXCR3 protein withIP-10.
 33. The antibody or functional antibody fragment of claim 32,wherein said antibody or fragment is monoclonal antibody 1C6 or anantigen binding fragment thereof.
 34. An antibody or antigen-bindingfragment thereof which binds a human CXCR3 protein.
 35. An antibody orantigen-binding fragment thereof of claim 34, wherein said binding canbe inhibited by a portion of a human CXCR3 corresponding to theN-terminal extracellular segment of SEQ ID NO:2 or a portion thereofhaving at least one immunological property of a human CXCR3 protein. 36.An antibody or antigen-binding fragment thereof of claim 34, whereinsaid binding can be inhibited by a polypeptide selected from the groupconsisting of a polypeptide having a sequence which is the same as thatof residues 1-15 of SEQ ID NO:2 and a polypeptide having a sequencewhich is the same as that of residues 16-30 of SEQ ID NO:2.
 37. A methodof detecting or identifying an agent which binds a mammalian CXCR3protein or ligand binding variant thereof, comprising combining an agentto be tested with a composition comprising an isolated and/orrecombinant mammalian CXCR3 Protein or ligand binding variant thereofunder conditions suitable for binding of ligand thereto, and detectingor measuring the formation of a complex between said agent and saidmammalian CXCR3 protein or variant.
 38. The method of claim 37, whereinthe agent is a ligand selected from the group consisting of human IP-10,human Mig, a mammalian homolog of IP-10, and a mammalian homolog of Mig.39. The method of claim 38, wherein the ligand is labeled with a labelselected from the group consisting of a radioisotope, spin label,antigen label, enzyme label flourescent group or chemiluminesent group.40. The method of claim 38, wherein the assay is a competition assay, inwhich binding is determined in the presence of one or more ligandsselected from the group consisting of human IP-10, human Mig, amammalian homolog of IP-10, and a mammalian homolog of Mig.
 41. A methodof detecting or identifying an agent which binds a mammalian CXCR3protein or a ligand binding variant thereof comprising: a) combining anagent to be tested with a host cell expressing recombinant mammalianCXCR3 protein or a ligand binding variant thereof under conditionssuitable for binding of ligand thereto; and b) detecting or measuringthe formation of a complex between said agent and the mammalian CXCR3protein or a ligand binding variant.
 42. The method of claim 41, whereinthe agent is a ligand selected from the group consisting of human IP-10,human Mig, a mammalian homolog of IP-10, and a mammalian homolog of Mig.43. The method of claim 41, wherein the assay is a competition assay, inwhich binding is determined in the presence of one or more ligandsselected from the group consisting of human IP-10, human Mig, amammalian homolog of IP-10, and a mammalian homolog of Mig.
 44. Themethod of claim 41, wherein the mammalian CXCR3 protein or a ligandbinding variant thereof can mediate cellular signalling and/or acellular response, and the formation of a complex is monitored bydetecting or measuring a signalling activity or cellular response ofsaid CXCR3 protein or variant in response thereto.
 45. A method ofdetecting or identifying an inhibitor of ligand binding to a mammalianCXCR3 protein or a ligand binding variant thereof comprising: a)combining an agent to be tested with a ligand of said mammalian CXCR3protein and a composition comprising isolated and/or recombinantmammalian CXCR3 protein or ligand binding variant thereof underconditions suitable for binding of ligand thereto; and b) detecting ormeasuring binding the formation of a complex between said mammalianCXCR3 protein or variant and said ligand, whereby inhibition of complexformation by the agent is indicative that the agent is an inhibitor. 46.The method of claim 45, wherein the ligand selected from the groupconsisting of human IP-10, human Mig, a mammalian homolog of IP-10, anda mammalian homolog of Mig.
 47. The method of claim 45, wherein thecomposition comprising isolated and/or recombinant mammalian CXCR3protein contains a host cell expressing recombinant mammalian CXCR3protein.
 48. The method of claim 47, wherein the mammalian CXCR3 proteincan mediate cellular signalling and/or a cellular response, and theformation of a complex is monitored by detecting or measuring asignalling activity or cellular response of said CXCR3 protein orvariant in response thereto.
 49. A method of detecting or identifying aninhibitor of ligand binding to a mammalian CXCR3 protein or ligandbinding variant thereof comprising: a) combining an agent to be testedwith a ligand of said mammalian CXCR3 protein and a host cell expressinga recombinant mammalian CXCR3 protein or ligand binding variant thereofunder conditions suitable for binding of ligand thereto; and b)detecting or measuring the formation of a complex between said proteinor variant and said ligand, whereby inhibition of complex formation bythe agent is indicative that the agent is an inhibitor.
 50. The methodof claim 49, wherein the ligand selected from the group consisting ofIP-10, Mig, a mammalian homolog of IP-10, and a mammalian homolog ofMig.
 51. The method of claim 49, wherein the mammalian CXCR3 protein canmediate cellular signalling and/or a cellular response, and theformation of a complex is monitored by detecting or measuring asignalling activity or cellular response of said CXCR3 protein inresponse thereto.
 52. The method of claim 49 wherein the agent is anantibody or antibody fragment.
 53. A method of detecting or identifyingan inhibitor of a mammalian CXCR3 protein or functional variant thereofcomprising combining an agent to be tested with (a) a host cellexpressing a recombinant mammalian CXCR3 protein or functional variantthereof, and (b) a ligand or promoter thereof, under conditions suitablefor detecting a ligand- or promoter-induced response, and assessing theability of the test agent to inhibit said response, whereby inhibitionof a ligand- or promoter-induced response by the agent is indicativethat the agent is an inhibitor.
 54. The method of claim 53, wherein theresponse is monitored by detecting or measuring a signalling activity orcellular response of said mammalian CXCR3 protein or variant thereof inresponse thereto.
 55. A method of detecting or identifying a promoter ofa mammalian CXCR3 protein of functional variant thereof comprisingcombining an agent to be tested with a host cell expressing arecombinant mammalian CXCR3 protein or functional variant thereof underconditions suitable for detecting a receptor-mediated response, anddetecting or measuring said response, whereby induction or stimulationof said response by the agent is indicative that the agent is apromoter.
 56. An inhibitor of at least one function characteristic of amammalian CXCR3 protein identified according to the method of claim 45.57. An inhibitor of claim 56, wherein the inhibitor is an antibody orportion thereof capable of binding receptor.
 58. An inhibitor of atleast one function characteristic of a mammalian CXCR3 proteinidentified according to the method of claim
 53. 59. A promoter of atleast one function characteristic of a mammalian CXCR3 proteinidentified according to the method of claim
 55. 60. A method ofdetecting a selected mammalian CXCR3 protein in a sample comprising: a)contacting a sample with an antibody which binds said protein underconditions suitable for specific binding of said antibody to theselected mammalian CXCR3 protein; and b) detecting antibody-CXCR3complexes.
 61. A method of modulating at least one function of amammalian CXCR3 protein, comprising the step of contacting said proteinwith an inhibitor or promoter of at least one function of said protein.62. A method for treating an inflammatory disease or condition,comprising administering to a mammal a therapeutically effective amountof an inhibitor of a mammalian CXCR3 protein, whereby inflammation isreduced.
 63. The method of claim 62, wherein the inflammatory disease orcondition is a T cell mediated disease or condition.
 64. The method ofclaim 62, wherein the inhibitor is an antibody or antigen-bindingfragment thereof which specifically binds a mammalian CXCR3 protein. 65.The method of claim 64, wherein the antibody or fragment thereof cancompete with monoclonal antibody 1C6 for binding to a human CXCR3protein.
 66. The method of claim 65, wherein the antibody or fragmentthereof is monoclonal antibody 1C6 or an antigen-binding fragmentthereof.
 67. A method of antitumor therapy, comprising administering toa mammal a therapeutically effective amount of a promoter of a mammalianCXCR3 protein, other than a natural ligand of said protein.
 68. A methodof antiviral therapy, comprising administering to a mammal atherapeutically effective amount of a promoter of a mammalian CXCR3protein, other than a natural ligand of said protein.
 69. Antisensenucleic acid comprising a nucleic acid having a sequence that hybridizesto a nucleic acid having the sequence: a) SEQ ID NO:1, b) a portionthereof comprising the coding sequence, c) or an RNA counterpart of anyone of (a) and (b).